Human growth in vitro


1Nuffield Department of Clinical Biochemistry, University of Oxford, John Radchffe Hospital, Headington, Oxford, UK 2Unileuer Research, Colworth House Sharnbrook, Bedford MK44 1LQ, UK * Present address: Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QR, UK

Summary We report for the first time the successful mainten- midine autoradiography confirmed that in vitro the ance and growth of hair follicles in vitro. in vivo pattern of DNA synthesis was maintained; Human anagen hair follicles were isolated by mic- furthermore, [35S]methionine labelling of rodissection from human . Isolation of the showed that their patterns of synthesis did not hair follicles was achieved by cutting the follicle at change with maintenance. the dermo-subcutaneous interface using a scal- The importance of this model to pel blade. Intact hair follicles were then removed is further demonstrated by the observations from the fat using watchmakers' forceps. that TGF-/51 has a negative growth-regulatory effect Isolated hair follicles maintained free-floating in on hair follicles in vitro and that EGF mimics the in supplemented Williams E medium in individual wells vivo depilatory effects that have been reported in of 24-well multiwell plates showed a significant sheep and mice. increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinised hair shaft, and was not associated with 3 Key words: human hair follicles, maintenance, growth the loss of hair follicle morphology. [methyl- H]ihy- factors.

Introduction Materials and methods The hair follicle is composed of epithelial components (the Materials matrix and outer ) and dermal components (the Williams E medium (minus glutamine), L-glutamine, Fungizone, dermal papilla and connective tissue sheath). Hair penicillin and streptomycin were supplied by Gibco, all other growth, which is effected by the division of the hair follicle tissue culture supplements came from Sigma. Polycarbonate matrix cells under control of the dermal papilla, is cyclical filters were supplied by the Nucleopore corporation. All radio- in the . Three distinct stages of hair growth can chemicals were from Amersham, GF/C filters came from be identified, an active phase (anagen) during which hair Whatman and ATP monitoring kits were supplied by LKB growth occurs, an intermediate regressive (catagen) stage Instruments Ltd. Mouse EGF and TPA were purchased from and a resting phase (telogen) during which no cell Sigma. Porcine TGF-/81 was from R&D systems Inc; synthetic proliferation occurs. The factors that regulate cell division human IGF-1 was supplied by Bachem Feinchemikalien. Minoxi- dil was a kind gift from Unilever Research, Colworth House, within the hair follicle matrix cells, and which control the Sharnbrook, Bedford; EGF, TGF-/81 and IGF-1 were all assayed hair growth cycle, are poorly understood, although growth for mitogenic activity using 3T3 or test cells at factors (Moore et al. 1981; Green et al. 1983; Green and Unilever and were found to have the expected biological Couchman, 1984; Panaretto et al. 1984; Nanney et al. 1984; activities. Akhurst etal. 1988; Green, 1989; Messenger, 1989), steroid hormones (Takayasu and Adachi, 1972; Schweikert and Wilson, 1974; Sultan et al. 1989), dermo-epithelial Isolation and maintenance of human hair follicles interactions (Jahoda et al. 1984) and the immune system Human anagen hair follicles were isolated by microdissection (Sawada et al. 1987; Pauss et al. 1989) have been from human scalp skin, taken from females aged 35-55 implicated. Our lack of understanding of the regulation of undergoing facelift surgery. Isolation of hair follicles was hair growth has been caused in part, by the lack of good in achieved by using a scalpel blade to cut through the skin at the vitro models (Philpott et al. 1989; Buhl et al. 1989). We now dermo-subcutaneous fat interface. The intact hair follicle bulb report for the first time on the successful growth of human was removed from the subcutaneous fat, under a stereo dissecting hair in vitro, and on the in vitro effects of growth factors microscope, using watchmakers' forceps, by gently gripping the and mitogens on our model. In particular, we show that of the follicle in the forceps and pulling the hair epidermal growth factor (EGF) mimics the in vivo follicle from the subcutaneous fat. This results in the isolation of depilatory action of EGF, resulting in the formation of a intact hair follicle bulbs without sustaining any visible damage, a club hair-like structure; and that transforming growth factor that is essential if successful maintenance of hair follicles is factor beta one (TGF-/31) may serve as a negative growth to be achieved. regulatory factor for the hair follicle. Isolated hair follicles were maintained in 500 [t\ of Williams E medium with supplements as previously described (Philpott et al. Journal of Cell Science 97, 463-471 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 463 1989). Follicles were maintained free-floating in individual wells 30min, following which 20/(1 of sample was removed and of 24-well multi-well plates, at 37 °C in an atmosphere of 5% neutralised with KOH. The sample was then centrifuged at CO2/95 % air. This permitted detailed measurements to be made 12 000 £ for 5min, after which a 10 /il sample was taken and on the length of individual hair follicles. Measurements were assayed for ATP by adding 8O/1I of 0.1M Tris-acetate buffer, made using a Nikon Diaphot inverted binocular microscope with pH 7.75, followed by 10 /il of monitoring reagent. The ATP content -piece measuring graticule. was then measured using a LKB 1250 luminometer.

Rates ofDNA and synthesis Autoradiography The rates of DNA and protein synthesis in isolated hair follicles Hair follicles were incubated for 6 h in 500 //I Williams E medium were investigated by measuring the rates of incorporation of containing 5 /(Ci [me£/ry/-3H]thymidine (spec. act. 3.3 /(Ci nmol"1). [mei/ty/-3H]thymidine and [U-14C]leucine, respectively, into per- After incubation follicles were washed in PBS supplemented with chloric acid (PCA)-precipitable material. Incubations were car- 10 mM thymidine and then fixed for 1 h in phosphate-buffered ried out in plastic Eppendorf tubes containing 500 /il of Williams formaldehyde. Follicles were then mounted in 3% agar fixed E medium supplemented with 1 ,«Ci of 3/IM [mekeratin synthesis plastic tubes containing 0.5 ml of distilled water in a gently These were investigated by incubating the hair follicles in 500 /il shaking water bath at 37 °C in an atmosphere of 5 % CO /95 % O . 35 2 2 Williams E medium containing 100 /(Ci of 1 mM [ S]methionine Incubations were carried out for 3 h. 1 (spec. act. 0.22/iCinmor ) for 24h at 37°C. Follicles were then After incubations were complete, the Eppendorf tubes contain- washed three times in PBS containing 10 mM methionine and ing the hair follicles were removed from their plastic tubes and then homogenised in ice-cold lysis buffer (1% Triton X-100, 1% briefly centrifuged at 12 000 g to bring down the hair follicles. The sodium deoxycholate, 0.1 % SDS, 50 mM NaCl, 5mM EDTA, 1 mM supernatant was then removed with a Pasteur pipette taking care phenylmethylsulphonyl fluoride (PMSF), 50 mM Tris-HCl, 30 mM not to remove the hair follicles as well. The follicles were then sodium pyrophosphate, pH7.4; Green et al. 1986). The homogen- washed by resuspending them in 1 ml of PBS supplemented with ate was centrifuged at 12 000 # in an Eppendorf microtube for 10 mM thymidine and 10 mM leucine; the follicles were then 15min and the supernatant was discarded. The pellet was then briefly centrifuged as before and the supernatant removed. After twice extracted with a high-salt buffer (600 mM KC1, 5 mM EDTA, three such washes the follicles were resuspended in 1 ml of 0.1 M 5mM EGTA, 50 mM Tris-HCl, pH7.4; Mischke and Wilde, 1987). EDTA, pH 12.3, and transferred using a Pasteur pipette to a The supernatant was discarded and the insoluble pellet analysed ground-glass homogeniser. Following homogenisation, the hom- by sodium dodecyl sulphate (SDS)-acrylamide gel electrophoresis ogenate was transferred to an Eppendorf tube and centrifuged for as described by Laemhi (1970). Gels were then dried under 15min at 12000^, to precipitate cell debris, after which the vacuum and autoradiographs produced using Kodak X-OMAT supernatant was removed for assay. Samples (100/d) were diagnostic film. removed for total DNA assay, and macromolecules in the Immunoblot analysis of to identify keratins was remaining supernatant were then precipitated by the addition of carried out by electrophoretic transfer from gels to nitrocellulose 500/d of 25 % (v/v) PCA. The samples were then left overnight at membranes (Towbin et al. 1979), using a Bio-Rad Trans Blot 4°C. system (Bio-Rad Laboratories). Membranes were incubated for The resulting precipitate was collected onto Whatman GF/C 2 h at room temperature in PBS containing 5 % skimmed milk, filters, under vacuum. The filters were then washed with 10 ml of 0.05% Tween 20 to reduce non-specific binding, and then 10 % (w/v) trichloroacetic acid (TCA) followed by 5 ml of 5 % (w/v) overnight with a wide spectrum rabbit anti-keratin polyclonal TCA and then dried with 1 ml of ethanol/diethyl ether (1:1, v/v). antibody (Dakopatts). The membrane was then washed for 30 min Radioactivity was counted in 10 ml of Optifluor® scintillant using in PBS containing 0.05 % Triton X-100, and then incubated for 2 h dual counting liquid scintillation spectrometry. Control exper- with a 1:1000 dilution of goat anti-rabbit IgG peroxidase iments were carried out in which the cell debris was solubilised in conjugate (Sigma). Bands were visualised by incubating the 1 ml of Soluene® and radioactivity counted in 10 ml of Optifluor® membranes with PBS containing O.Smgml"1 3,3'-diaminobenzi- as described above, these controls showed that less than 10 % of dine, 0.01 % H O . incorporated radioactivity was discarded in the cell debris pellet. 2 2

Hair follicle DNA content Results Total hair follicle DNA content was determined using the fluorometric diaminobenzoic acid method (DABA) of Fiszer- Isolation and maintenance of hair follicles Szafarz et al. (1981). Portions (100/il) of sample were freeze dried under vacuum using a Virtis unitrap vacuum freeze drier. DABA It was found that by cutting into thin strips (0.45gml~') was made up in distilled water decolorised by approximately 3-5 mm x 10 mm and then using a scalpel shaking with activated charcoal (0.15 g ml"1) for 3h, then filtered blade to cut away the subcutaneous fat at the level of the through a Millipore filter (0.45 Jim) and stored in a glass vial in , and using a pair of watchmakers' forceps, the dark until required (DABA was prepared fresh daily and not it was possible to isolate in excess of 100 human anagen stored overnight). DABA (100 /il) was then added to the samples, hair follicles in 1-2 h from apiece of skin 4 cm x 2 cm. Hair which were incubated in a water bath for 45min at 60°C. After follicles in the early catagen stage of their growth cycle this 1.5 ml of 1M HC1 was added to the samples and the were occasionally seen, but were not used in these fluorescence measured using a Perkin Elmer LS5 spectrofluor- ometer with an excitation wavelength of 420 nm and an emission experiments. wavelength of 520 nm. DNA standards were made up in 0.1M Measurements made on freshly isolated human hair EDTA and treated in exactly the same way as the samples. follicles and at 24 h intervals show (Fig. 1) that in vitro isolated human hair follicles significantly increased in Hair follicle ATP contents length over 4 days in culture (P<0.001); the rate of growth This was measured using LKB-ATP monitoring kits based on the in vitro being 0.3 mm a day (n=6 patients, 36 follicles in luciferin luciferase assay of ATP (Stanley and Williams, 1969). total), which approximates closely to that seen in vivo Hair follicles were placed in 500 jil of Williams E medium to which (Myers and Hamilton. 1951). was added 100 j.A of 20 % PCA; the follicles were then left on ice for Photographs taken of freshly isolated and maintained 464 M. P. Philpott, M. R. Green and T. Kealey The pattern of keratin synthesis was studied by incubating hair follicles with [35S]methionine for 24 h at 37 °C, after which cell extracts were separated by one- dimensional SDS-PAGE and analysed by autoradiogra- phy. The pattern of keratin synthesis observed in freshly isolated hair follicles under our conditions is shown in Fig. 4A (lane 1). In freshly isolated hair follicles using a 10 % gel we were able to resolve five major bands; a doublet 3 of 56 and 59K (K=10 Mr) and a triplet of 48/49/50K. We also observed a number of faint lower molecular weight bands between 40 and 46K, which included a doublet at 44/46K. Control experiments using immunoblot analysis of gels with a wide-spectrum keratin antibody confirmed that all these bands were keratins (Fig. 4B). It was also observed that the pattern of keratin synthesis remained unchanged in hair follicles main- tained for 4 days (Fig. 4A, lane 2).

0.0 f 1 1 1 • i I i 1 1 ' 1 i The effects of growth factors and mitogens on human 0 24 72 96 hair follicle growth in vitro and on the rates of /methyl- 48 3 14 Time (h) H]thymidine and [U- C]leucine uptake and on hair follicle ATP content Fig. 1. Human hair follicle growth in vitro. Graph shows The results of this study are shown in Table 1. All human hair follicle growth in culture over 96 h. Results measurements of hair follicle length were carried out over expressed as the mean±s.E.M. for sequential measurements a 72 h period and rates of [me£/iy/-3H]thymidine and [U- made on hair follicles isolated from «=6 skin biopsies 14C]leucine uptake and hair follicle ATP content were (minimum of 6 hair follicles used from each biopsy). measured after 72 h in culture. Table 1 shows that for hair follicles maintained for 72 h hair follicles (Fig. 2) show that the increase in length over in Williams E medium containing 1 % FCS the rate of hair 4 days was not associated with any disruption of hair follicle growth was 0.81 ±0.04 mm per 72 h, the rate of follicle architecture. In particular, the length increase can [me£/iyZ- HJthymidine uptake was 2.57±0.35pmol,t/g~1 be seen from Fig. 2 to be attributed to the production of a DNA per 3h (mean±s.E.M.), and the rate of [U- C]leucine keratinised hair shaft. uptake 204±24pmol//g~1 DNA per 3h (mean±s.E.M.)- In order to determine whether the increase in hair hair follicle ATP content was 713±65pmolfollicle"1 follicle length observed above was due to the normal (mean±s.E.M.). mechanisms of cell proliferation and migration, [methyl- When hair follicles were maintained with EGF 3H]thymidine autoradiography was carried out. Fig. 3A (lOngml"1) the rate of hair follicle growth was shows that in the freshly isolated follicles the typical 0.70±0.05mm per 72 h and was not significantly different pattern of DNA synthesis is taking place, with the from that of follicles maintained in 1 % FCS alone, but majority of thymidine uptake occurring in the matrix cells there was a most striking change in the morphology of of the hair follicle bulb, adjacent to the dermal papilla. hair follicles maintained with lOngml"1 EGF. Fig. 5 Autoradiography carried out on hair follicles maintained shows sequentially at 24 h intervals the changes that for 4 days (Fig. 3B) shows that over 4 days of maintenance occur in the overall morphology of human hair follicles this pattern remains constant. maintained with EGF (lOngml"1). These observations

Table 1. The effects of growth factors on in vitro hair follicle growth, [methy\-3 HJthymidine and [U-14C]leucine uptake and hair follicle ATP content Follicle growth [3HJthymidine [14C]leucine ATP content (mm over 72 h) (pmol/ig^'DNA per 3h) (pmol;ig"1DNA per 3h) (pmol follicle"1) Treatment (n=6 samples) (n=6 samples) (n=6 samples) (n=3 samples) 1 % FCS 0.81±0.04 2 57±0 35 204 ±24 713±65 TGF-/31 (lOngmP1) 0.57+0.03"* 1.56±0.21* 175±22 653±20 IGF-1 OOngml-1) 0.76±0.05 4 04±0 39* 255±30 485±39* EGF (lOngml"1) 0.70±0.05 1.19±0 37* 357 ±71 698±43 TPAdOOngml"1) 0.46±0 04*** 1.29±0.29* 165±28 297±16* 20 % FCS 0.85±0.05 2.64±0.37 328±42 Not measured Serum-free 0.93±0.03* 2.71±0.64 193 ±28 610±54 Minoxidil 200 /ig ml"1 0.63±0.05* 1.78±0.27 169±21 694 ±28 10/igml"1 0.83±0.03 3.51±0.62 232 ±14 630 ±52 200neml~1 0.91 ±0.03 2.47±0.70 148 ±43 618±98 Hair follicles were isolated and maintained in Williams E medium containing supplements as described in the text, with the additional growth- regulatory factors as listed in the table. Hair follicle measurements were made on at least 6 hair follicles from each sample of skin. Rates of [methyl- 3HJthymidine and lU-14CJleucine uptake were measured after the hair follicles had been maintained for 72 h in the presence of the relevant growth factors. Experiments were carried out using 5 follicles in duplicate from each skin sample. Hair follicle ATP contents likewise were measured after hair follicles had been maintained for 72 h with the relevant growth factor using 4 follicles in duplicate from each sample. Statistical analysis was carried out using Student's /-test to compare differences between follicles maintained with 1% FCS and treated follicles (*/><0.05. **P<0 01,

Human hair growth in vitro 465 2A


Fig. 2. Light micrographs taken under an inverted microscope showing the sequential growth of the same hair follicle in culture over 96 h. Clearly visible are the dermal papilla (dp), hair follicle matrix (m), outer root sheath (ors), connective tissue sheath (cts) and hair shaft (h). The increase in hair follicle length can be seen to be attributed to the production of a hair shaft. (A) Freshly isolated hair follicle; (B) after 24 h maintenance; (C) 48 h; (D) 72 h, and (E) 96 h.

show that over a 72 h period, human hair follicles per 3h (mean±s.E.M.), but did significantly reduce the maintained with lOngml"1 EGF show considerable ATP content of the hair follicle to 485±39pmol follicle"1 changes in their hair follicle morphology, especially in the (mean±S.E.M., P<0.05). hair follicle bulb where the hair shaft forms a club hair- For hair follicles maintained with 12-O-tetra-decanoyl- like structure. This structure then moves slowly upwards phorbol-13-acetate (TPA, lOOngml"1) the rate of growth within the hair follicle over 72 h until after 5 days it is was reduced to 0.46±0.04mm per 72 h; this value was nearly extruded from the hair follicle. There was moreover significantly less (P<0.001) than that of hair follicles a significant decrease in the rate of [me£/ry/-3H]thymidine maintained with 1 % FCS alone. The rate of [methyl- uptake to 1.19±0.37pmolfig"1 DNA per 3h (mean± 3H]thymidine uptake for hair follicles was S.E.M.) when compared with that for follicles maintained 1.29±0.29pmol,i/g~1 DNA per 3h (mean±s.E.M.) and this in 1% FCS, the rate of [U-14C]leucine uptake at value too was significantly less than that for hair follicles 357±71pmol,i/g~1 DNA per 3h, and a hair follicle ATP maintained in 1% FCS alone (P<0.05). TPA had no content of 698±32pmol follicle"1 were, however, not significant effect on the rate of [U-14C]leucine uptake, significantly different. which was 165±28pmol;/g~1 DNA per 3h (mean±s.E.M.), but did significantly reduce the hair follicle ATP content to In the presence of TGF-/31 the rate of increase of hair 1 follicle length was found to be 0.57±0.03mm per 72 h 297± 16 pmol follicle" . (mean±S.E.M.); this was significantly less (P<0.001) than For hair follicles maintained in serum-free Williams E that of hair follicles maintained with 1 % FCS. The rate of medium the rate of hair follicle growth was 0.93 ±0.03 mm [me£/yZ-3H]thymidine uptake for hair follicles maintained per 72 h (mean±s.E.M.), this value was significantly with TGF-/31 was l.setO^lpmol/zg"1 DNA per 3h higher than that for follicles maintained in Williams E (mean±s.E.M.), which was also significantly less than that medium containing 1% FCS (P<0.05). There was no of follicles maintained with 1 % FCS (P<0.05). The rate of significant difference between the rates of [methyl- [U-14C]leucine uptake at 175±22pmol,ug"1 DNA per 3h 3H]thymidine or [U-14C]leucine uptake in follicles main- was not, however, significantly different, and neither was tained in serum-free medium when compared with those the hair follicle ATP content at 653±20pmol follicle"1. maintained in medium containing 1 % FCS, nor was there IGF-1 (SOngml"1) had no significant effect on hair any significant difference between the ATP contents of follicle length in vitro, but did significantly increase hair follicles maintained in serum-free medium or 1 % (P<0.05) the rate of [metfi;yZ-3H]thymidine uptake to FCS. For hair follicles maintained in 20 % FCS there was 1 also no significant difference in the rate of hair follicle 4.04±0.39pmolf(g~ DNA per 3h (mean±s.E.M.). IGF-1 3 did not, however, have any significant effect on the rates of elongation and the rates of [me£/i;yZ- H]thymidine uptake 14 1 14 [U- C]leucine uptake, which was 255±30pmol1«g~ DNA and [U- C]leucine uptake or hair follicle ATP contents. 466 M, P. Philpott, M. R. Green and T. Kealey m


Fig. 3. Tritiated thymidine autoradiographs of isolated human hair follicles showing: (A) freshly isolated; and (B) 96 h maintained follicles. Freshly isolated hair follicles show the typical pattern of DNA synthesis in the hair follicle with the majority of thymidine uptake occurring in the matrix cells (m) of the hair follicle bulb, adjacent to the dermal papilla (dp). After 96 h in culture the pattern of DNA synthesis remains unchanged.

Experiments carried out on hair follicles maintained follicle growth after 5 days in culture (P<0.01) and with Minoxidil at 200 ng ml"1 or at lOjUgml"1 showed that (P<0.001), respectively, when compared to control exper- neither of these had any significant effect on either the iments. EGF (lOngml"1) and IGF-1 OOngml"1) had no rates of hair follicle growth over 72 h or on the rates of significant effect on hair follicle length when compared to [methyl-3Withymidme uptake or [U-14C]leucine uptake, controls. nor was there any significant effect on hair follicle ATP The effects of Minoxidil on hair follicles maintained for 5 content. For hair follicles maintained with Minoxidil at days is shown in Fig. 8 and shows that 200ngml~1 200,ugml~1 for 72 h there was a significant inhibition Minoxidil appeared to stimulate hair follicle growth (P<0.05) of the rate hair follicle growth to 0.63±0.05mm significantly over 5 days (P<0.01) when compared with per 72 h (mean±s.E.M.); Minoxidil at this concentration controls. Minoxidil at lO.t/gml"1 had no significant effect had no significant effect on the rate of either [methyl- on hair follicle growth; whereas 200 //g ml"1 Minoxidil 3H]thymidine or [U-14C]leucine uptake, or on the hair significantly inhibited hair follicle growth (P<0.01). follicle ATP content. Discussion The effects of growth factors and mitogens on the longer- term growth of human hair follicles in vitro In this study we have demonstrated for the first time the The effects of serum on the longer-term growth of hair successful maintenance and growth of human hair follicles follicles in vitro is shown in Fig. 6 and shows that serum in vitro. We have shown that human hair follicles isolated has an inhibitory effect on hair follicles maintained over a by microdissection and maintained free-floating show a 5-day period. It was observed that at 5 days hair follicles significant increase in hair follicle length (P<0.01) over 4 maintained in serum-free medium were still growing in a days in culture when maintained in Williams E medium linear fashion; but that hair follicles maintained with 1 % containing 1 % FCS. The rate of increase in hair follicle FCS were significantly inhibited (P<0.01) as was the length was 0.3 mm/day, and this rate of increase approxi- growth of hair follicles maintained in 20% FCS (P<0.01). mates to that seen in the in vivo scalp hair follicle (Myers There was no significant difference between hair follicles and Hamilton, 1951). It was observed that this increase in maintained in 1 % and 20 % FCS. hair follicle length was not associated with any loss of hair The effects of growth factors and TPA are shown in follicle architecture and, furthermore, it was observed that Fig. 7. This figure shows that both TGF-/31 (lOngml"1) the increase in hair follicle length was associated with an and TPA (lOOngml"1) significantly inhibited human hair increase in the length of the keratinised hair shaft.

Human hair growth in vitro 467 x10 -3






-24 -33

-24 -16 Fig. 4. (A) [35S]methionine autoradiography showing the pattern of keratin synthesis in freshly isolated hair follicles (lane 1) and after 96 h maintenance (lane 2), and showing that the pattern of keratin synthesis remains -16 unchanged in maintained hair follicles. All B gels were loaded with the total keratin extract from five hair follicles. (B) Immunoblot carried out using a broad spectrum anti-keratin antibody on freshly isolated hair follicles (lane 1) and after 96 h maintenance (lane 2). Confirming that the bands observed in A are keratins.

Further evidence to support the successful maintenance the overall patterns remain unchanged and support our of hair follicles in vitro is demonstrated by [methyl- other observations, and also show that we are able to 3H]thymidine autoradiography. In freshly isolated hair maintain human hair follicles in vitro successfully, and follicles the majority of DNA synthesis takes place in the that they continue to produce a keratinised hair shaft. matrix cells of the hair follicle bulb. It was observed that To demonstrate further the importance of this model we in hair follicles maintained for 4 days this pattern of have studied the effects of a number of growth-regulatory synthesis was maintained. These data show that in vitro factors and mitogens on the in vitro rates of hair follicle hair follicles are able to maintain the in vivo pattern of elongation, [me£/iyZ-3H]thymidine and [U-14C]leucine up- DNA synthesis, and so it is reasonable to suppose that the take and on hair follicle viability as determined by production of a keratinised hair shaft in the maintained measuring their ATP contents. The most dramatic effects hair follicle occurs as a result of matrix cell division in the were observed when the hair follicles were maintained hair follicle bulb. with EGF (10 ngml"1). EGF receptors are found on rat and We have also observed that the pattern of keratin human hair follicles (Moore et al. 1981; Green et al. 1983), synthesis observed in freshly isolated hair follicles is and in vivo experiments on mice show that EGF appears to sustained in hair follicles maintained in vitro for 4 days. act as a specific inhibitor of matrix cell division (Moore et These observations on the patterns of keratin synthesis al. 1981). In sheep, EGF acts as a depilatory by inducing a are, however, only a preliminary study, and to character- premature anagen-to-catagen transformation. As a result ise fully the patterns of keratin synthesis would require of this the hair fibre is weakened and this permits two-dimensional gels. However, these data confirm that shearing (Panaretto et al. 1984).

468 M. P. Philpott, M. R. Green and T. Kealey 5A


Fig. 5. Light micrograph taken under an inverted microscope showing the sequential effects of epidermal growth factor on the same hair follicle maintained in culture over a 72 h period: (A) freshly isolated hair follicle; (B) after 24 h in culture; (C) 48 h; (D) 72 h, showing the formation of a club hair-like structure, which apparently migrates upwards in the hair follicle, and resembles the in vivo depilatory action of EGF. The hair follicle shown in this figure was isolated from a dark-haired individual and shows a highly pigmented region adjacent to the dermal papilla, this contrasts with the hair follicle shown in Fig. 2, which was taken from a fair-haired individual and as such is not so highly pigmented. However, the EGF effect is the same in both pigmented and non-pigmented hair follicles.

In our model we have shown that isolated human hair 3H]thymidine uptake in response to lOngml 1 TGF-/31. follicles maintained with EGF show considerable morpho- However, this is not accompanied by the gross morphologi- logical changes. We have found that EGF promotes the cal alterations observed with EGF. The TGF-/31 effect in formation of a club hair-like structure, which appears to vitro is curious because, although TGF-/3 has been shown migrate upwards in the hair follicle until by day 5 it is to be a potent inhibitor of proliferation of epithelial cells in virtually extruded from the hair-follicle. This in vitro vitro (Sporn et al. 1987), it is known that topical TPA depilatory effect, which appears to mimic the in vivo action stimulates mouse hair growth (Ogawa and Hattori, 1983; of EGF, confirms the value of this model in hair-follicle Inohara et al. 1988), and it has also been shown that biology, and may also point to a possible role for EGF in topical TPA promotes the expression of TGF-/J mRNA in regulating anagen to catagen transformation during the vivo (Akhurst et al. 1988). However, we show in Table 1 hair growth cycle. Table 1 shows that in the presence of that, in vitro, TPA also inhibits hair follicle growth. This is EGF the hair follicles remain viable as determined by both compatible with our in vitro TGF-/51 effect, and indicates the hair follicle ATP content and [U-14C]leucine uptake; that in vivo there may be further uncharacterised dermal however, as expected, the rates of [me^yZ-3H]thymidine paracrine phenomena or dose-response effects. uptake are significantly reduced. Dermal fibroblasts produce IGF-1 in vitro (Messenger, Immunohistochemistry has shown that TGF-/31 is 1989); however, in our model we found no increase in hair present in the mammalian dermal papilla (Heine et al. follicle length in vitro, despite the significant stimulation 1987) although the biological activity of this form is not of [methyl- H]thymidine uptake. However, it is possible clear. Its in vivo function in the hair follicle is also not that the insulin present in our supplemented culture known; however, we have now shown that in vitro TGF-/31 medium is saturating the IGF-1 receptors, although this inhibits hair growth. Table 1 shows a significant reduction would not explain the observed stimulation of [methyl-3H]- in both the rates of hair follicle lengthening and [methyl- thymidine uptake.

Human hair growth in vitro 469 48 72 96 120 Time in culture (h)

24 48 72 96 Fig. 7. Graph showing the effects of growth factors and Time in culture (h) mitogens on isolated human hair follicles maintained in vitro over 5 days in the presence of 1 % FCS, EGF (lOngmP1), IGF- Fig. 6. Graph showing the effects of serum on isolated human 1 OOngmr^/rGF-zSl (lOngml-1) and TPA (lOO/igmP1). hair follicles maintained in vitro over 5 days. Results are Results are expressed as the mean±s.E.M. for sequential expressed as the mean±s.E.M. for sequential measurements measurements made on hair follicles isolated from n=6 skin made on hair follicles isolated from n=6 skin biopsies (6 hair biopsies (6 hair follicles used from each biopsy, 36 hair follicles follicles used from each biopsy, 36 hair follicles in total). in total). (•) 1% FCS; (O) IGF-1; (•) EGF; (•) TGF-/81; (•) Serum-free; (O) 1 % FCS; (•) 20 % FCS. (•) TPA.

In this study we have also looked at the effect of serum on maintained human hair follicles and shown that when presence of Minoxidil we observed that 200ngml 1 human hair follicles were maintained in serum-free Minoxidil (0.95 JUM) significantly stimulated hair follicle medium they were still growing in a linear fashion after 5 growth. This was not apparently due, however, to an days in culture whereas hair follicles maintained in tissue actual stimulation of hair growth, it appeared that the culture medium containing 1% FCS were significantly Minoxidil was counteracting the serum-induced cessation inhibited after 5 days in culture (P<0.01), as were hair of hair growth at 4 days. This model, therefore, may be follicles maintained in 20% FCS (P<0.01); there was no useful for dissecting Minoxidil's mode of action. The significant difference between hair follicles maintained in concentration of Minoxidil that was active in our 1 % or 20 % FCS. These observations indicate that human contrasts with those of Buhl et al. (1989) and Waldon et al. hair follicles maintained in culture do not apparently have (1989), who have reported, using a rat vibrissae culture a requirement for serum for elongation and that serum system, that the minimum effective dose of Minoxidil is factors may in fact be inhibitory. It is known that TGF-/3 is between 0.5 HIM and 1.0 HIM. In the human hair follicle we present in serum (Childs et al. 1982) and as we have have found that lO/ugml"1 (48,UM) Minoxidil had no already shown in this study TGF-/3 is a potent inhibitor of significant effect on human hair follicles whereas hair follicle growth in vitro. It will be interesting to see 200,ugml~1 (0.95mM) significantly inhibited hair follicle whether it is TGF-/3 in the serum that is inhibiting hair growth. Again these observations contrast with those of follicle growth or whether other inhibiting factors are Buhl et al. (1989), who report that only a concentration as present. high as 10 mM Minoxidil is inhibitory. Clearly, human Minoxidil stimulates human hair growth in vivo hair follicles isolated by the methods described here are (Clissold and , 1987). We found, however (Table 1), much more sensitive to Minoxidil. that up to 72 h in vitro Minoxidil either had no effect, or at In conclusion, we have developed an in vitro model for 200/(g ml"1 significantly inhibited hair growth, without human hair growth that reproduces the in vivo rate of hair effect on cell viability. Recent in vivo studies, however growth, as well as the apparent in vivo pattern of cell (Frienkel et al. 1989), indicate that Minoxidil may not division in the hair follicle matrix cells. The importance of increase the rate of hair growth as such, but rather it may this model in hair follicle biology has been demonstrated increase the length of anagen by shortening the time that by the in vitro effects of EGF, which mimic those seen in the hair follicle is in the resting stage of its growth cycle. vivo, and by the growth inhibitory effects of TGF-/31. Also, We would, therefore, not necessarily have expected a the observation that hair follicles grow for a longer period Minoxidil stimulation of an anagen hair follicle that was of time in serum-free medium suggests that they are able already growing at a rate close to that seen in vivo. to regulate their own growth, possibly by the production of However, for hair follicles maintained for 5 days in the relevant growth regulatory factors. This should prove

470 M. P. Philpott, M. R. Green and T. Kealey • 200ngml Minoxidil GREEN, M. R., CLAY, C. S., GIBSON, W. T , HUGHES, T. C, SMITH, C. G., WESTOATE, G. E. AND KEALEY, T. (1986). Rapid isolation in large 0 10//gml Minoxidil numbers of intact, viable individual hair follicles from the skin: Biochemical and ultrastructural characterisation. J invest. Derm. 87, 768-770. #200;

Human hair growth in vitro 471