British Journal of Dermatology (1992) 127, 600-607.
Rat hair follicle growth in vitro M.P.PHILPOTT, M.R.GREEN* AND T.KEALEY Department of Clinical Biochemistry. University of Cambridge, Addenhrookes Hospital, Hills Road, Cambridge CB2 2QR, U.K. 'Unilever Research, Colworth House. Sharnbrook, Bedford MK44 ll.Q, U.K. Accepted for publication 9 )une 1992
Summary
Pelage hair follicles were isolated by gentle microdissection from 8-12-day-old rats, and maintained in supplemented Williams E medium. Length measurements made on freshly isolated hair follicles, and at 24-h intervals, showed a signiticant increase in hair follicle length over 48 h, after which time no further significant increase in length was observed. Photomicrographs of maintained follicles showed that this increase in hair follicle length could be attributed to the production ofa keratinized hair shaft. Histology and [methyl-*H] thymidine autoradiography of freshly isolated hair follicles showed the dermal papilla to be elongated, with thymidine uptake located predominantly in the matrix cells ofthe hair follicle bulb adjacent to the dermal papilla. This pattern remained unaltered for the first 48 h of maintenance, but after 72 h the dermal papilla had rounded into a tight ball of cells, with very little thymidine uptake occurring in the adjacent matrix cells. On maintenance, fetal calf serum (FCS). epidermal growth factor (EGF) and 12-o-tetradecanoyl phorbol 1 S-acetate (TPA) all significantly stimulated [methyl-'H] thymidine and [IJ-'^C| leucine uptake, but inhibited hair follicle elongation. Insulin-like growth factor-1 (IGF-1) had no significant effect on rates of hair follicle elongation and [methyl-'H] thymidine uptake, but significantly stimulated rates of [D-^^C] leucine uptake. Transforming growth factor-/?l (TGF-^1) significantly inhibited both the rate of [methyl-^H] thymidine uptake and hair foUicle elongation.
The in vitro growth ofhair follicles over a 4-5 day period in skin plugs taken from post-embryonic mouse skin has been reported.' However, these studies did not permit detailed measurements to be made on individual hair follicles. Attempts at culturing individual hair follicles from post-embryonic mouse-' and rat skin^ have met with little success. We have shown that rat hair follicles isolated by 'shearing'^ appear to be viable and intact on isolation, but when maintained in vitro on permeable supports fail to grow.*" We recently reported the successful growth of isolated human hair follicles in vitro.' showing that by careful isolation methods it is possible to obtain undamaged human hair follicles which grow in vitro at an in vivo rate. This present study describes the in vitro growth of rat hair follicles isolated by an improved gentle microdissection technique. The effects of EGF. TGF-/J1. IGF-1, and TPA on the rate of hair follicle elongation were examined, together with their eff'ects on the rates of fmethyl-'H] thymidine and [II-'^^C] leucine uptake and on hair follicle viability as assessed by their adenosine triphosphate (ATP) content.
Correspondence: Dr M.P.Philpott.
600
Methods Materials Williams E medium, L-glutamine, Fungizone®. penicillin/streptomycin and colchicine were supplied by Gibco. All other tissue culture supplements and mouse EGF and TPA were from Sigma. All radiochemicals were supplied by Amersham: ATP monitoring kits were supplied by Pharmacia LKB. Porcine TGF-^1 was from R & D Systems Inc.. and synthetic human IGF-l was supplied by Bachem Feinchemikalien. Isolation of rat hair follicles by microdissection Rat hair follicles were isolated by a simple microdissection method. Strips of dorsal skin (2-5 x 0- 5 cm) were removed from 8-12-day-old Wistar rats. These strips were cut into small pieces, approximately 0-5 cm-, and individual pieces were placed in a Petri dish containing isolation medium (PBS/EBSS. 1:1). The skin was gently torn into small strips under a binocular microscope using watch-maker's forceps. Hair follicles were then isolated from the skin by gentle manipulation with the watch-maker's forceps. Isolated hair follicles were
RAT HAIR FOLLICLE GROWTH IN VITRO
601
immediately transferred to experimental media as described below. Hair follicle maintenance
(i) Elongation measurements. Individual, freshly isolated hair follicles were maintained at J7°C in an atmosphere of 5% CO2/95% air in separate wells of 96-well plates containing 1 50/d of Williams E medium with 1%FCS, 2 mM L-glutamine. insulin (10/jg/ml).transferrin (10/ig/ ml), hydrocortisone (10 ng/mi), sodium selenite (10 ng/ ml). Fungizone® (2-5 |ig/ml). penicillin/streptomycin (100 ll/ml: 100 //g/ml| and trace element mix (Gibco), Measurements were made using a Nikon Diaphot inverted microscope equipped with an eyepiece measuring graticule. (ii) Organ maintenance. Organ maintenance of freshly isolated rat hair follicles was carried out using four-well plates. Each well contained 4 ml of Williams E medium supplemented as described above. Hair follicles were maintained free-floating, with 20 follicles in each well, in an atmosphere of 5% CO2/95% air, at 37°C. Rates of DNA and protein synthesis
These were investigated by measuring the rates of incorporation of [methyl-'H] thymidine and [U-"'C] leucine, respectively, into perchloric acid (PCA) precipitable material, as previously described.' Incubations were carried out in plastic Eppendorf tubes containing SOO /(I of Williams E medium supplemented with 1 /iCi of 5 fiM [methyl-'H] thymidine (specific activity 0 67 mCi/ ^M) and 0 5 /tCi of 0 5 mM [U-'-'C] leucine (specific activity 2 mCi/miw). Follicles in Eppendorf tubes were incubated in stoppered plastic tubes containing 0- 5 ml of distilled water in a gently shaking water bath at 3 7°C in an atmosphere of 5% CO2/9 5% O2 for 3 h. Follicles were then washed three times with PBS supplemented with 10 mM thymidine and 10 mM leucine, and then homogenized in 1ml of 0-1 M E D T A , pH 12-3. This was then centrifuged for 15 min at 12,000 g to precipitate
Figure 2, Light micrograph ofthc bulb region of an isolalt'd rat hair follicle at higher power, showing the dermal papilla (DPI. matrix (M). outer root sheath (ORS| and hair shaft (HS) (scale bar = 2()0 nm).
ORS
Figure 1. Light micrograph of a freshly isolated hair follicle from a 10day-old rat showing hair follicle bulb with dermal papilla IDP|. hair shaft (HS) and outer root sheath (ORS) (scale bar= 200 fim).
cell debris, and the supernatant was removed for assay. Aliquots (100 jul) were removed for total DNA assay.^ Macromolecules in the remaining supernatant were precipitated by the addition of 500/^1 of 25% (v/v) PCA, and the resulting precipitate was collected on to Whatman GF/C filters, under vacuum. Filters were washed with 10 ml of 10% (w/v) trichloroacetic acid (TCA) followed by 5 ml of 5% (w/v) TCA, and dried with 1 ml of ethanol/diethyl ether (1:1). Radioactivity was counted in 10 ml of Optitluor® scintillant. using dual counting liquid scintillation spectrometry.
Autoradiographfj
This was carried out as described previously.' Briefly, hair follicles were incubated for 6 h with 5 f.iCi of [methyl-'H] thymidine. Following incubation, the follicles were washed three times with PBS supplemented with 10 mM thymidine. and fixed for 1 h with 3%
602
M.P.PHILPOTT et al
0.7 _
00
Time in culture (h) Figure 3. Changes in rat hair follicle length with time in culture. Experiments were carried out as described in the text. Results are expressed as the mean ±SEM for n = 6 rats (six follicles from each rat, J6 follicles in total). Statistical analysis of data was carried out using Student's I-test. • . control; O, colchicine.
glutaraldehyde. Follicles were then mounted in 5% agar to assist handling during subsequent processing. The agar blocks were fixed overnight in 3% glutaraldehyde, then washed in PBS and processed into Historesin. Sections {15 /im) were cut, and autoradiography was carried out using K5 dipping emulsion (Ilford). Hair follicle ATP content
This was measured using LKB-ATP monitoring kits based on the luciferin/luciferase assay of ATP.'^ Hair follicles were placed in 500 /xl of Williams E medium, to which was added 100 /il of 20% PCA. Twenty microlitres of sample was removed and neutralized with 1 M KOH. Aliquots {10/tl) were made up to 90/il with 0 1 M Tris-Acetate buffer, pH 7-75, and the ATP content measured using LKB-ATP monitoring reagent and a LKB 1250 luminometer.
Results It was found that, by cutting rat skin into thin strips, large numbers of intact individual hair follicles could be
Figure 4. Light micrographs showing increase in hair follicle length with time in culture, (a I Freshly isolated, (b| after 24 h in culture, [c) after 48 h in culture. Showing that the increase in hair follicle length can be attributed to the production of a keratinized hair shaft, although the freshly produced hair fibre appears thinner than the original (arrows) (scale bar = 2()()/im|.
isolated. This was achieved by gently grasping the hair follicles in the strips with watch-maker's forceps, as close to the dermal/subcutaneous fat interface as possible. By gently pulling downward, it was found that the follicles could easily be removed from the skin (Fig, 1), When observed under an inverted binocular microscope at higher power {Fig. 2). hair follicles showed an apparently intact morphology. When maintained free-floating in individual wells of 96-well plates, they showed a significant increase in length over 48 h in culture {0-24 mm/day, Pj vitro. These data are consistent with the conclusion that isolated rat hair follicles can show true hair growth in vitro for at least 48 h, and that these changes are not due to swelling or other artefactual changes. After 48 h in culture, it would appear that, although DNA synthesis in the matrix cells ofthe hair follicles stops, these cells continue to differentiate, giving rise to the formation of a keratinized hair follicle bulb. It is not clear why rat hair follicles do not continue to grow for more than 48 h in culture, when human follicles can be maintained for at least 9 days.^ The rat hair follicles do not appear to be damaged on isolation, as far as may be determined by light microscopy. However, rat hair follicles isolated from 8-12-day-old rats are much smaller than human hair follicles, and they may be exposed to greater trauma on isolation than human hair follicles. It is well known that when hair follicles are stressed they respond by premature entry into eatagen.'" Moreover, as the first hair growth cycle of the rat is very short, and these follicles would normally enter eatagen
604
M.P.PHILPOTT et al.
Figurefi. Autoradiography. showing the patterns of I methyl-'H I thymidine uptake in: (a) freshly isolated rat hair follicles, with the majority of uptake in the matrix cell region of the hair follicle adjacent to the dermal papilla |DP); (b| after 48 h in culture the dermal papilla remains elongated, and the majority of uptake still occurs in the adjacent matrix cells: (c) after 72 h in culture considerable changes have taken place in the hair follicle bulb; the dermal papilla has formed a rounded structure, and very little uptake takes place in the adjacent matrix cells (scale bar= 100 ;jm).
RAT HAIR FOLLICLE GROWTH IN VITRO
605
06_
0,8
-S
0.6.
Ji p
0.4
.s u
0.0
02.
Time inctilture (h) 00
24
48
72
Time iti culture (h) Kigure 7. The effects of serum nn the length of isolated rat hair lollicles maintained in vitro. Experiments were carried out as described in the text. Results arc expressed as the mean±SRM for n^b rats (h follicles from each rat. JCi follicles in totall. Statistical analysis of data was carried out using Student's I-test, • , serum free; O 1% FCS: • , 20% FCS,
at around 1 7 days of age.'' it is possible that as a result of the trauma of isolation they are more likely to enter a catagen-Iike state prematurely. It would therefore be interesting to discover whether hair follicles isolated from older rats in either the third or fourth hair growth cycle, in which the follicles have a more prolonged anagen phase, can be maintained for longer. It has also been shown that steroid hormones regulate the activity of rat pelage follicles,'^ and the sudden change in hormonal environment in culture may have more effect on rat pelage follicles than human scalp hair follicles. Increasing concentrations of FCS were found to significantly stimulate both [methyl-^H] thymidine uptake and [U-''^C] leucine uptake, but hair follicles grew significantly longer in serum-free medium than the follicles maintained in either 1 or 20% FCS; this is similar to our observations on isolated human hair follicles, in which we found that serum significantly inhibited hair follicle growth in vitro.''^ It was observed that, when rat hair follicles were
I-'igure 8. The effects of growth factors and mitogen.s on the length of isolated rat hair follicles maintained in vitro. Experiments were carried out as described in the text. Results are expressed a.s the mean ± SEM for n=h rats (J6 follicles in total). Statistical analysis of data was carried out using Student's f-test, D. control; • , [(;[-'-l;C3, TGE-/(I: • . EGF; O. TPA.
maintained in vitro with FCS. cells explanted from the ORS region ofthe hair follicle, whereas this did not occur in hair follicles maintained in serum-free medium. Thus, serum seems to contain factors that stimulate ORS cell division, but inhibit matrix cell division. These results suggest that in isolated rat hair follicles FCS is able to stimulate ORS cell division, but to specifically inhibit the matrix cells which are responsible for hair follicle growth. These factors have yet to be fully characterized, although TGF-j3 is known to be present.'* In this study we have shown that, when freshly isolated rat hair follicles are maintained in vitro in the presence of 10 ng/ ml TCF-jil. hair follicle elongation was significantly inhibited, but ATP content remained unchanged, suggesting that the observed inhibition of hair follicle growth was not due to a cytotoxic effect, Interestingly. TGF-^. which inhibits the growth of human epidermal keratinocytes in vitro, does not inhibit their protein synthesis over 3 days.''^ We observed the same uncoupling in the hair follicle, in that TGF-^ inhibited [methyl'H] thymidine uptake but not [U-^"^C1 leucine uptake. These observations are similar to those we reported for isolated human hair follicles.' and indicate that in the hair follicle TGF-/n may act as both a negative growth regulator and a promoter of hair follicle keratinocyte maturation.
606
M.P.PHILPOTT et ai
Treatment Serum free 1%FCS 10% FCS 20% FCS EGF TGF-^1 IGF-1 TPA
[methyl-'H|thymidinc pmoi//igDNA/5h
[U-'Y] ieucine pmol/;igDNA/ih
1.29±O,2] (6) 1-22±O'14(I9) l - 9 4 ± ( ) i 2 (6)
196±29|6) 156±15 119) J19±107(6)
2-57±O-24tnil2| 2'12±O'17§§§(16| O'48±O'O8§§§19) 1.66±0.24(10) 2'5J±O'25§§§(4)
258±19§§§(16) 133±23 (9) 276±45§(1O) 533±1O4§(4)
J10±44tt(12)
Hair follicle ATP pmol/20 follicles 291 ± 3 6 141 250±27(4) Not measured 347±f)8 (41 285±94|4| 243±16(4) 224±12(4) 432±48§§(4)
Table 1. The effects of growth factors and mitogens on the rates of |mcthyl-'H) thymidine and [U-'^C] leucine uptake and hair follicle ATP content of isolated rat hair hair follicles maintained for 24 h. Numbers in parentheses indicate number rats used for each observation
Fjcperiments were performed as described in the texl using 20 follicles in duplicate from each rat. Statistical analysis of data was carried out using Student's (-test, llndicates values significantly different from serum free medium (|P
Rat hair follicle growth in vitro M.P.PHILPOTT, M.R.GREEN* AND T.KEALEY Department of Clinical Biochemistry. University of Cambridge, Addenhrookes Hospital, Hills Road, Cambridge CB2 2QR, U.K. 'Unilever Research, Colworth House. Sharnbrook, Bedford MK44 ll.Q, U.K. Accepted for publication 9 )une 1992
Summary
Pelage hair follicles were isolated by gentle microdissection from 8-12-day-old rats, and maintained in supplemented Williams E medium. Length measurements made on freshly isolated hair follicles, and at 24-h intervals, showed a signiticant increase in hair follicle length over 48 h, after which time no further significant increase in length was observed. Photomicrographs of maintained follicles showed that this increase in hair follicle length could be attributed to the production ofa keratinized hair shaft. Histology and [methyl-*H] thymidine autoradiography of freshly isolated hair follicles showed the dermal papilla to be elongated, with thymidine uptake located predominantly in the matrix cells ofthe hair follicle bulb adjacent to the dermal papilla. This pattern remained unaltered for the first 48 h of maintenance, but after 72 h the dermal papilla had rounded into a tight ball of cells, with very little thymidine uptake occurring in the adjacent matrix cells. On maintenance, fetal calf serum (FCS). epidermal growth factor (EGF) and 12-o-tetradecanoyl phorbol 1 S-acetate (TPA) all significantly stimulated [methyl-'H] thymidine and [IJ-'^C| leucine uptake, but inhibited hair follicle elongation. Insulin-like growth factor-1 (IGF-1) had no significant effect on rates of hair follicle elongation and [methyl-'H] thymidine uptake, but significantly stimulated rates of [D-^^C] leucine uptake. Transforming growth factor-/?l (TGF-^1) significantly inhibited both the rate of [methyl-^H] thymidine uptake and hair foUicle elongation.
The in vitro growth ofhair follicles over a 4-5 day period in skin plugs taken from post-embryonic mouse skin has been reported.' However, these studies did not permit detailed measurements to be made on individual hair follicles. Attempts at culturing individual hair follicles from post-embryonic mouse-' and rat skin^ have met with little success. We have shown that rat hair follicles isolated by 'shearing'^ appear to be viable and intact on isolation, but when maintained in vitro on permeable supports fail to grow.*" We recently reported the successful growth of isolated human hair follicles in vitro.' showing that by careful isolation methods it is possible to obtain undamaged human hair follicles which grow in vitro at an in vivo rate. This present study describes the in vitro growth of rat hair follicles isolated by an improved gentle microdissection technique. The effects of EGF. TGF-/J1. IGF-1, and TPA on the rate of hair follicle elongation were examined, together with their eff'ects on the rates of fmethyl-'H] thymidine and [II-'^^C] leucine uptake and on hair follicle viability as assessed by their adenosine triphosphate (ATP) content.
Correspondence: Dr M.P.Philpott.
600
Methods Materials Williams E medium, L-glutamine, Fungizone®. penicillin/streptomycin and colchicine were supplied by Gibco. All other tissue culture supplements and mouse EGF and TPA were from Sigma. All radiochemicals were supplied by Amersham: ATP monitoring kits were supplied by Pharmacia LKB. Porcine TGF-^1 was from R & D Systems Inc.. and synthetic human IGF-l was supplied by Bachem Feinchemikalien. Isolation of rat hair follicles by microdissection Rat hair follicles were isolated by a simple microdissection method. Strips of dorsal skin (2-5 x 0- 5 cm) were removed from 8-12-day-old Wistar rats. These strips were cut into small pieces, approximately 0-5 cm-, and individual pieces were placed in a Petri dish containing isolation medium (PBS/EBSS. 1:1). The skin was gently torn into small strips under a binocular microscope using watch-maker's forceps. Hair follicles were then isolated from the skin by gentle manipulation with the watch-maker's forceps. Isolated hair follicles were
RAT HAIR FOLLICLE GROWTH IN VITRO
601
immediately transferred to experimental media as described below. Hair follicle maintenance
(i) Elongation measurements. Individual, freshly isolated hair follicles were maintained at J7°C in an atmosphere of 5% CO2/95% air in separate wells of 96-well plates containing 1 50/d of Williams E medium with 1%FCS, 2 mM L-glutamine. insulin (10/jg/ml).transferrin (10/ig/ ml), hydrocortisone (10 ng/mi), sodium selenite (10 ng/ ml). Fungizone® (2-5 |ig/ml). penicillin/streptomycin (100 ll/ml: 100 //g/ml| and trace element mix (Gibco), Measurements were made using a Nikon Diaphot inverted microscope equipped with an eyepiece measuring graticule. (ii) Organ maintenance. Organ maintenance of freshly isolated rat hair follicles was carried out using four-well plates. Each well contained 4 ml of Williams E medium supplemented as described above. Hair follicles were maintained free-floating, with 20 follicles in each well, in an atmosphere of 5% CO2/95% air, at 37°C. Rates of DNA and protein synthesis
These were investigated by measuring the rates of incorporation of [methyl-'H] thymidine and [U-"'C] leucine, respectively, into perchloric acid (PCA) precipitable material, as previously described.' Incubations were carried out in plastic Eppendorf tubes containing SOO /(I of Williams E medium supplemented with 1 /iCi of 5 fiM [methyl-'H] thymidine (specific activity 0 67 mCi/ ^M) and 0 5 /tCi of 0 5 mM [U-'-'C] leucine (specific activity 2 mCi/miw). Follicles in Eppendorf tubes were incubated in stoppered plastic tubes containing 0- 5 ml of distilled water in a gently shaking water bath at 3 7°C in an atmosphere of 5% CO2/9 5% O2 for 3 h. Follicles were then washed three times with PBS supplemented with 10 mM thymidine and 10 mM leucine, and then homogenized in 1ml of 0-1 M E D T A , pH 12-3. This was then centrifuged for 15 min at 12,000 g to precipitate
Figure 2, Light micrograph ofthc bulb region of an isolalt'd rat hair follicle at higher power, showing the dermal papilla (DPI. matrix (M). outer root sheath (ORS| and hair shaft (HS) (scale bar = 2()0 nm).
ORS
Figure 1. Light micrograph of a freshly isolated hair follicle from a 10day-old rat showing hair follicle bulb with dermal papilla IDP|. hair shaft (HS) and outer root sheath (ORS) (scale bar= 200 fim).
cell debris, and the supernatant was removed for assay. Aliquots (100 jul) were removed for total DNA assay.^ Macromolecules in the remaining supernatant were precipitated by the addition of 500/^1 of 25% (v/v) PCA, and the resulting precipitate was collected on to Whatman GF/C filters, under vacuum. Filters were washed with 10 ml of 10% (w/v) trichloroacetic acid (TCA) followed by 5 ml of 5% (w/v) TCA, and dried with 1 ml of ethanol/diethyl ether (1:1). Radioactivity was counted in 10 ml of Optitluor® scintillant. using dual counting liquid scintillation spectrometry.
Autoradiographfj
This was carried out as described previously.' Briefly, hair follicles were incubated for 6 h with 5 f.iCi of [methyl-'H] thymidine. Following incubation, the follicles were washed three times with PBS supplemented with 10 mM thymidine. and fixed for 1 h with 3%
602
M.P.PHILPOTT et al
0.7 _
00
Time in culture (h) Figure 3. Changes in rat hair follicle length with time in culture. Experiments were carried out as described in the text. Results are expressed as the mean ±SEM for n = 6 rats (six follicles from each rat, J6 follicles in total). Statistical analysis of data was carried out using Student's I-test. • . control; O, colchicine.
glutaraldehyde. Follicles were then mounted in 5% agar to assist handling during subsequent processing. The agar blocks were fixed overnight in 3% glutaraldehyde, then washed in PBS and processed into Historesin. Sections {15 /im) were cut, and autoradiography was carried out using K5 dipping emulsion (Ilford). Hair follicle ATP content
This was measured using LKB-ATP monitoring kits based on the luciferin/luciferase assay of ATP.'^ Hair follicles were placed in 500 /xl of Williams E medium, to which was added 100 /il of 20% PCA. Twenty microlitres of sample was removed and neutralized with 1 M KOH. Aliquots {10/tl) were made up to 90/il with 0 1 M Tris-Acetate buffer, pH 7-75, and the ATP content measured using LKB-ATP monitoring reagent and a LKB 1250 luminometer.
Results It was found that, by cutting rat skin into thin strips, large numbers of intact individual hair follicles could be
Figure 4. Light micrographs showing increase in hair follicle length with time in culture, (a I Freshly isolated, (b| after 24 h in culture, [c) after 48 h in culture. Showing that the increase in hair follicle length can be attributed to the production of a keratinized hair shaft, although the freshly produced hair fibre appears thinner than the original (arrows) (scale bar = 2()()/im|.
isolated. This was achieved by gently grasping the hair follicles in the strips with watch-maker's forceps, as close to the dermal/subcutaneous fat interface as possible. By gently pulling downward, it was found that the follicles could easily be removed from the skin (Fig, 1), When observed under an inverted binocular microscope at higher power {Fig. 2). hair follicles showed an apparently intact morphology. When maintained free-floating in individual wells of 96-well plates, they showed a significant increase in length over 48 h in culture {0-24 mm/day, Pj vitro. These data are consistent with the conclusion that isolated rat hair follicles can show true hair growth in vitro for at least 48 h, and that these changes are not due to swelling or other artefactual changes. After 48 h in culture, it would appear that, although DNA synthesis in the matrix cells ofthe hair follicles stops, these cells continue to differentiate, giving rise to the formation of a keratinized hair follicle bulb. It is not clear why rat hair follicles do not continue to grow for more than 48 h in culture, when human follicles can be maintained for at least 9 days.^ The rat hair follicles do not appear to be damaged on isolation, as far as may be determined by light microscopy. However, rat hair follicles isolated from 8-12-day-old rats are much smaller than human hair follicles, and they may be exposed to greater trauma on isolation than human hair follicles. It is well known that when hair follicles are stressed they respond by premature entry into eatagen.'" Moreover, as the first hair growth cycle of the rat is very short, and these follicles would normally enter eatagen
604
M.P.PHILPOTT et al.
Figurefi. Autoradiography. showing the patterns of I methyl-'H I thymidine uptake in: (a) freshly isolated rat hair follicles, with the majority of uptake in the matrix cell region of the hair follicle adjacent to the dermal papilla |DP); (b| after 48 h in culture the dermal papilla remains elongated, and the majority of uptake still occurs in the adjacent matrix cells: (c) after 72 h in culture considerable changes have taken place in the hair follicle bulb; the dermal papilla has formed a rounded structure, and very little uptake takes place in the adjacent matrix cells (scale bar= 100 ;jm).
RAT HAIR FOLLICLE GROWTH IN VITRO
605
06_
0,8
-S
0.6.
Ji p
0.4
.s u
0.0
02.
Time inctilture (h) 00
24
48
72
Time iti culture (h) Kigure 7. The effects of serum nn the length of isolated rat hair lollicles maintained in vitro. Experiments were carried out as described in the text. Results arc expressed as the mean±SRM for n^b rats (h follicles from each rat. JCi follicles in totall. Statistical analysis of data was carried out using Student's I-test, • , serum free; O 1% FCS: • , 20% FCS,
at around 1 7 days of age.'' it is possible that as a result of the trauma of isolation they are more likely to enter a catagen-Iike state prematurely. It would therefore be interesting to discover whether hair follicles isolated from older rats in either the third or fourth hair growth cycle, in which the follicles have a more prolonged anagen phase, can be maintained for longer. It has also been shown that steroid hormones regulate the activity of rat pelage follicles,'^ and the sudden change in hormonal environment in culture may have more effect on rat pelage follicles than human scalp hair follicles. Increasing concentrations of FCS were found to significantly stimulate both [methyl-^H] thymidine uptake and [U-''^C] leucine uptake, but hair follicles grew significantly longer in serum-free medium than the follicles maintained in either 1 or 20% FCS; this is similar to our observations on isolated human hair follicles, in which we found that serum significantly inhibited hair follicle growth in vitro.''^ It was observed that, when rat hair follicles were
I-'igure 8. The effects of growth factors and mitogen.s on the length of isolated rat hair follicles maintained in vitro. Experiments were carried out as described in the text. Results are expressed a.s the mean ± SEM for n=h rats (J6 follicles in total). Statistical analysis of data was carried out using Student's f-test, D. control; • , [(;[-'-l;C3, TGE-/(I: • . EGF; O. TPA.
maintained in vitro with FCS. cells explanted from the ORS region ofthe hair follicle, whereas this did not occur in hair follicles maintained in serum-free medium. Thus, serum seems to contain factors that stimulate ORS cell division, but inhibit matrix cell division. These results suggest that in isolated rat hair follicles FCS is able to stimulate ORS cell division, but to specifically inhibit the matrix cells which are responsible for hair follicle growth. These factors have yet to be fully characterized, although TGF-j3 is known to be present.'* In this study we have shown that, when freshly isolated rat hair follicles are maintained in vitro in the presence of 10 ng/ ml TCF-jil. hair follicle elongation was significantly inhibited, but ATP content remained unchanged, suggesting that the observed inhibition of hair follicle growth was not due to a cytotoxic effect, Interestingly. TGF-^. which inhibits the growth of human epidermal keratinocytes in vitro, does not inhibit their protein synthesis over 3 days.''^ We observed the same uncoupling in the hair follicle, in that TGF-^ inhibited [methyl'H] thymidine uptake but not [U-^"^C1 leucine uptake. These observations are similar to those we reported for isolated human hair follicles.' and indicate that in the hair follicle TGF-/n may act as both a negative growth regulator and a promoter of hair follicle keratinocyte maturation.
606
M.P.PHILPOTT et ai
Treatment Serum free 1%FCS 10% FCS 20% FCS EGF TGF-^1 IGF-1 TPA
[methyl-'H|thymidinc pmoi//igDNA/5h
[U-'Y] ieucine pmol/;igDNA/ih
1.29±O,2] (6) 1-22±O'14(I9) l - 9 4 ± ( ) i 2 (6)
196±29|6) 156±15 119) J19±107(6)
2-57±O-24tnil2| 2'12±O'17§§§(16| O'48±O'O8§§§19) 1.66±0.24(10) 2'5J±O'25§§§(4)
258±19§§§(16) 133±23 (9) 276±45§(1O) 533±1O4§(4)
J10±44tt(12)
Hair follicle ATP pmol/20 follicles 291 ± 3 6 141 250±27(4) Not measured 347±f)8 (41 285±94|4| 243±16(4) 224±12(4) 432±48§§(4)
Table 1. The effects of growth factors and mitogens on the rates of |mcthyl-'H) thymidine and [U-'^C] leucine uptake and hair follicle ATP content of isolated rat hair hair follicles maintained for 24 h. Numbers in parentheses indicate number rats used for each observation
Fjcperiments were performed as described in the texl using 20 follicles in duplicate from each rat. Statistical analysis of data was carried out using Student's (-test, llndicates values significantly different from serum free medium (|P
