The FASEB Journal express article 10.1096/fj.03-0244fje. Published online December 4, 2003.

Thymosin 4 increases hair growth by activation of hair

follicle stem cells
Deborah Philp, Mychi Nguyen, Brooke Scheremeta, Sharleen St-Surin, Ana M. Villa,
Adam Orgel, Hynda K. Kleinman, and Michael Elkin
Cell Biology Section, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892
Corresponding author: Hynda K. Kleinman, Ph.D., Cell Biology Section, NIH, NIDCR, Building
30, Room 433, 30 Convent Dr. MSC 4370, Bethesda, MD 20892. E-mail:
hkleinman@dir.nidcr.nih.gov
ABSTRACT
Thymosin 4, a 43-amino acid polypeptide that is an important mediator of cell migration and
differentiation, also promotes angiogenesis and wound healing. Here, we report that thymosin 4
stimulates hair growth in normal rats and mice. A specific subset of hair follicular keratinocytes
in mouse skin expresses thymosin 4 in a highly coordinated manner during the hair growth
cycle. These keratinocytes originate in the hair follicle bulge region, a niche for skin stem cells.
Rat vibrissa follicle clonogenic keratinocytes, closely related, if not identical, to the bulgeresiding stem cells, were isolated and their migration and differentiation increased in the
presence of nanomolar concentrations of thymosin 4. Expression and secretion of the
extracellular matrix-degrading enzyme matrix metalloproteinase-2 were increased by thymosin
4. Thus, thymosin 4 accelerates hair growth, in part, due to its effect on critical events in the
active phase of the hair follicle cycle, including promoting the migration of stem cells and their
immediate progeny to the base of the follicle, differentiation, and extracellular matrix
remodeling.
Key words: matrix metalloproteinase-2 clonogenic keratinocytes

he mature hair follicle is a complex miniorgan that has a tightly regulated growth cycle.
During postnatal development, the follicle undergoes successive phases of active hair
shaft production (anagen), apoptosis-driven regression (catagen), and a quiescent phase
(telogen) (1). During the anagen phase, active hair growth involves cell proliferation in the
proximal follicular epithelium, followed by invasion of the elongating follicle into the
subcutaneous tissue, differentiation of the epithelium at the base of the follicle, and formation of
hair matrix cells, which proliferate and generate a new hair shaft. Then a regression phase
(catagen) of the hair growth cycle ensues, during which the lower part of the follicle undergoes
programmed cell death and involution (13). At this point, the follicle enters telogen, the resting
period. The cycle is then repeated. The ability of hair follicles to constantly renew is ensured by
the presence of the multipotent stem cells, which, upon division, generate two types of daughter
cells. Some of the daughter cells retain the same multipotent phenotype, while others become
rapidly dividing transit-amplifying (TA) cells, which provide differentiated progeny for the

regeneration of the lower follicle at the onset of each new cycle (4, 57). The bulge region of the
follicle, located close to the insertion of the arrector pili muscle, has been identified as a stem
cell niche (510). At the onset of anagen, bulge-localized, multipotent stem cells or their
daughter TA cells migrate to the base of the follicle to further differentiate to matrix cells and to
produce a new hair shaft (6, 7, 9, 10). Interestingly, cells emanating from the bulge region
migrate downward to repopulate the hair matrix and also migrate upwards to replenish the skin
epithelium and may therefore contribute to wound healing processes (9).
Thymosin 4, a ubiquitous 4.9-kDa polypeptide originally isolated from bovine thymus, is a
potent mediator of cell migration and differentiation (1116). It was identified as a gene upregulated four- to sixfold during early endothelial cell tube formation and found to promote
angiogenesis. It is present in wound fluid (17), and when added topically or given systemically, it
promotes angiogenesis and wound healing (13). Thymosin 4 elicits cell migration through a
specific interaction with actin (18, 19). Recently, we demonstrated that a central 7-amino acid
(LKKTETQ) actin binding domain has both angiogenic and wound healing activity while other
domains are inactive (20). In angiogenesis and in wound healing, thymosin 4 acts by
accelerating the migration of endothelial cells and keratinocytes and increasing the production of
extracellular matrix-degrading enzymes (14, 19). Thymosin 4 also has anti-inflammatory
activity (11, 21).
The process of hair growth utilizes many cellular and molecular mechanisms common to
angiogenesis and wound healing, namely migration, differentiation, and remodeling of the
extracellular matrix (911, 2225). In the present study, we investigated the role of thymosin 4
in hair growth in different in vitro and in vivo experimental models. Thymosin 4 promotes hair
growth in normal rats and mice. A specific subset of follicular keratinocytes in the mouse skin,
which originates at the bulge region, expresses thymosin 4. The temporal and spatial
distribution of these keratinocytes parallel the pattern reported for the stem cells and their
daughter TA cells at the different stages of the hair cycle (9, 10). We isolated clonogenic
keratinocytes from the bulge compartment of the rat vibrissa follicle, further characterized them
as an immediate progeny of the stem cells, and found that these cells express high levels of
thymosin 4 when cultured in vitro. We show that thymosin 4 promotes hair clonogenic
keratinocyte cell migration, as well as secretion of the extracellular matrix-degrading enzyme
matrix metalloproteinase 2 (MMP-2). We also found that thymosin 4 initiated early
differentiation of these cells based on reduction in the expression of keratin 15, a specific marker
of epidermal stem cells (26). Taken together, our results suggest that in addition to its known
angiogenic and wound healing effects, thymosin 4 is a naturally occurring modulator of hair
growth that acts by stimulation of stem cell migration, protease production, and differentiation.
METHODS
Hair growth
Thymosin 4 was prepared by the FDA (Bethesda, MD). Thymosin 4 and the peptides at 0.05%
(w/v) in 0.2% polyacrylic acid hydrogel (from Mahnaz Badamchian, George Washington
University School of Medicine) were applied topically to the dorsal lateral areas of shaved rats.
For the rat studies, thymosin 4 (0.05%) was applied to a dorsal/lateral area of the skin and the

control vehicle was applied to an opposing adjacent area on the same animal (3-5 rats/dose). For
the mouse studies, 8-wk-old wild-type C57BL6 mice (2 mice/dose) with their hair in telogen
phase, as identified by their pink backskin color (27), were shaved and treated separately with
either thymosin 4 (0.02%) or control vehicle. The skin samples were fixed in 4%
paraformaldehyde, embedded in paraffin, and 5 m sections were stained with MassonTriChrome. The number of hair follicles in the histological sections was counted by two different
blinded observers with at least three sections of different fields counted per rat (3-5 rats/data
point). These experiments were repeated twice with similar results. Microphotos were taken at
x32 for the rat skin and x66 for the mouse skin with a Zeiss Stem SVII dissecting scope.
Induction of hair cycle
Depilation was used to induce hair growth in resting follicles, as described previously by Paus et
al. (28). The dorsal skin of 8-wk-old female C57BL/6 mice at the telogen phase (as identified by
their pink skin color) was depilated using hair remover wax strip kit (Del Laboratories,
Farmingdale, NY), leading to synchronized development of anagen hair follicles. Skin tissue
samples were collected at day 0 (telogen), day 4 postdepilation (early anagen), and day 9
postdepilation (late anagen; 5 mice/time point). The samples were fixed in 4% paraformaldehyde
and processed for histological examination and immunostaining. This experiment was done
twice with similar results.
Immunohistochemistry
Immunohistochemical staining was performed on 5 m paraffin sections using a polyclonal
rabbit antibody raised against full-length thymosin 4 peptide sequence (from Allan Goldstein,
George Washington University School of Medicine). Primary antibody was detected using Dako
EnVision Kit (DakoCytomation, Denmark), and counterstaining was performed using
hematoxylin.
Immunofluorescence
After culture on glass coverslips (12 mm; Carolina Biological Supply Company, NC), cells were
fixed with 4% paraformaldehyde in PBS with 5% sucrose. The cells were incubated with
antibody raised against thymosin 4 peptide, followed by staining with secondary CY3conjugated donkey anti-rabbit antibody, diluted 1:200. Samples were mounted with
GEL/MOUNTTM (Biomeda Corp., Foster City, CA) and visualized with a Zeiss LM 510
confocal microscope.
Isolation of clonogenic keratinocytes (hair follicle stem cells)
Clonogenic keratinocytes were isolated from Fisher 344 rat vibrissa follicles as described
previously by Kobayashi et al. (29). After the animals were killed, the upper lip containing the
vibrissal pad was cut, and its inner surface was exposed. While the animals were under a
dissecting microscope, vibrissa follicles were dissected and plucked from the pad. A fragment of
the follicle containing the bulge region was cut off and incubated for 30 min in
collagenase/dispase solution (1 mg/ml; Roche Molecular Biochemicals) at 37C. The epithelial
core was detached from the collagen capsule and further incubated in 0.05%

trypsin/collagenase/dispase solution (30 min, 37C) to facilitate the dissociation of epithelial

cells. Isolated cells were cultured in keratinocyte-SFM medium, containing EGF (2.5 g/500
ml), bovine pituitary extract (25 mg/500 ml; Invitrogen, Carlsbad, CA), and 10% FCS. The
seeding density was 1000 cells/35 mm plate, and 60-80 colonies per plate were formed. For the
proliferation assay, 5000 cells were seeded per well in 96-well plates, and proliferation was
assessed using the CellTiter AQueous Cell Proliferation Assay Kit (Promega, Madison, WI).
This experiment was repeated three times with similar results.
Western blot
Cells were lysed by addition of RIPA buffer, and equal protein aliquots of cell lysates were
separated on 4-12% Bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA). Proteins were transferred
to a nitrocellulose membrane (Invitrogen) and detected using polyclonal antibodies against
mouse keratins (Covance Research Products, Richmond, CA). K15 antibodies were obtained
from Covance and NeoMarkers (Fremont CA). The membranes were stripped and reprobed with
anti-GAPDH antibody (Research Diagnostics, Flanders, NJ), and densitometry measurements
were taken using NIH Image Software with keratin 15 normalized using GAPDH.
Migration assays
Migration was studied in 48-well Boyden chambers using 8-m pore polycarbonate, PVPF,
membranes (Poretics, Livermore, CA) coated with 50 g/ml of collagen type 1 (BD Biosciences,
Bedford, MA) diluted in keratinocyte-SFM that contained 72 mM HEPES buffer. Cultured
clonogenic keratinocytes were harvested using trypsin and resuspended in keratinocyte-SFM
containing 1% bovine serum albumin factor-V and 25 mM HEPES-buffer. The bottom chamber
was filled with increasing amounts of thymosin 4.. Fibroblast-conditioned medium was the
positive control. Keratinocytes, 30,000 cells/well, were added to the upper chambers and were
incubated at 37C with 5% CO2 for 4.5 h. The membranes were then fixed and stained with DiffQuik (VWR, Bridgeport, NJ). Cell migration was quantitated in three random microscopic fields
of triplicate wells. Cells were acquired at x10 magnification using a Nikon Optiphot-2
microscope for counting. The assay was repeated twice.
Motility
Clonogenic keratinocytes were plated on 35-mm dishes. Migration was monitored for 20 h using
a Zeiss inverted microscope. Digital images were collected using a CCD camera (model 2400;
Hamamatsu Photonics) at 10-min intervals, stored as image stacks, converted to QuickTime
movies, and analyzed using MetaMorph Group 3.5 software (Universal Imaging Corp., London,
UK). This experiment was repeated twice, and six cells were tracked in each experiment.
Zymography
Clonogenic keratinocytes were cultured for 9 days. After 16 h serum deprivation, the cells were
incubated for 6 h in the presence of increasing concentrations of thymosin 4. Aliquots of the cell
lysate and resulting conditioned medium were analyzed for gelatinolytic activity, using Novex
Zymogram Gels (Invitrogen). The band intensity was determined by densitometry measurements
using NIH Image Software.

Statistics
The InStat program was used to determine P values. All data are means SD.
RESULTS
Thymosin 4 promotes hair growth in rats and mice
While studying wound healing in rat skin, we unexpectedly observed visually and at the
histological level increased hair growth at the wound margins 7 days after topical treatment with
thymosin 4 (unpublished observation). In this study, we have shaved the skin of healthy rats and
applied thymosin 4 topically on one side of the shaved area and the control vehicle on the
opposing lateral side of the same animal. After 7 days of treatment, we observed an increased
number of anagen-phase hair follicles in the skin areas treated with thymosin 4 (Fig. 1a and d).
The number of anagen follicles was approximately twofold greater than in rats treated with
vehicle alone. The increased number of hairs in anagen phase was retained with continued triweekly treatment over 30 days. Within 14 days of treatment cessation, the number of active hair
follicles decreased to control levels. We next tested whether thymosin 4 would promote hair
growth in 8-wk-old C57BL6 wild-type mice. Animals used in this experiment have all of their
hair follicles in the telogen stage as judged by their pink skin color (27). The mice were shaved
and thymosin 4 was applied topically on the shaved area as described in Methods. Control
animals were treated with vehicle alone. As shown in Fig. 1c and f, thymosin 4-treated (but not
control) animals displayed quick hair regrowth. Histological examination confirmed the
thymosin 4-induced activation of the hair follicles (Fig. 1b and e).
Thymosin 4 protein expression by a subset of hair follicle stem cells
We first explored the spatial and temporal pattern of endogenous thymosin 4 expression in hair
follicles during depilation-induced, synchronized adult hair cycling in C57BL/6J mice. We
wanted to correlate the observed effects of thymosin 4 administration with possible functional
involvement of endogenous thymosin 4 in hair growth. Low levels of thymosin 4 protein were
observed in follicles at the telogen (resting) phase, before depilation (Fig. 2). In these follicles,
thymosin 4 expression was confined to a small number of cells residing in the bulge region, at
the level of the insertion of the arrector pili muscle. Hair follicle transition to early anagen (day 4
after depilation) was associated with an increased number of thymosin 4-expressing cells in the
bulge region (Fig. 2). Moreover, some thymosin 4-positive-stained cells were detected in the
lower part of the follicle, between the bulge and bulb area (Fig. 2, arrowhead). At late anagen
(day 9 after depilation), a significant number of the cells located in the lower follicle (matrixsurrounding part of the outer root sheath) expressed thymosin 4 both in the nucleus and
cytoplasm. The sebaceous gland was stained at all stages, due to nonspecific absorption as found
by others with several different antibodies. Thus, with the progression of the hair growth cycle,
thymosin 4-positive cells, initially detected only in the bulge, were observed at the bulb area,
suggesting that they are migrating from the bulge region. These data show that the temporal and
spatial distribution of thymosin 4-expressing cells was similar to the pattern proposed for the
hair follicle stem cells and their daughter TA cells, i.e., emanating from the bulge and migrating
downward to give rise to matrix cells that subsequently generate the hair shaft (9, 10).

Cultured rat vibrissa clonogenic keratinocytes express thymosin 4

We studied rat vibrissae follicle keratinocytes from the bulge region, representing the stem cell
population (10, 30), to determine if isolated stem cells express thymosin 4. Previously, hair
follicle stem cells have been identified as bulge-residing keratinocytes with a high in vitro
clonogenic potential (58, 10, 29-31). Although hair follicle stem cells are not fully characterized
in terms of specific markers, they preferentially express keratin 15 (K15) (26). We isolated
clonogenic keratinocytes from the rat vibrissa bulge region and found that the isolated cells were
highly clonogenic (Fig. 3a). These cells were positive for the stem cell marker keratin 15 as well
as for keratins 5, 6, and 14 (Fig. 3c), also known to be expressed by bulge stem cells (32).
Furthermore, these cells lacked keratin 10 (Fig. 3c), a known early marker of terminal
keratinocyte differentiation (4, 31). Moreover, when cultured in vitro, these cells were able to
move with an average velocity of 0.43 m/min, a typical mobility rate reported for epidermal
stem cells and their daughter TA cells (33). Based on these characteristics and on previous
reports by Kobayashi et al., (29) and Oshima et al., (10), we conclude that the obtained cell
population represents the immediate progeny of hair follicle stem cells. Using RT-PCR and
immunofuorescent staining approaches, we found that these cells expressed thymosin 4 mRNA
(not shown) and protein (Fig. 3b) after 7-10 days of culturing in vitro. Interestingly, treatment of
the clonogenic keratinocytes with exogenous thymosin 4 caused a dose-dependent decrease in
the expression levels of the multipotent undifferentiated stem cell marker K15 (Fig. 4). A decline
in K15 is associated with stem cell differentiation. Further, we found that thymosin 4 had no
effect on stem cell proliferation (data not shown). These data indicate that the clonogenic
keratinocytes isolated from rat vibrissa bulge represent the stem cell population and suggest that
thymosin 4 is important for early stem cell differentiation.
Thymosin 4 promotes the migration of hair clonogenic keratinocytes in vitro
Thymosin 4 has been previously shown to promote endothelial cell migration (14). Here, we
found that cultured clonogenic keratinocytes migrate to thymosin 4 after 4.5 h in Boyden
chamber assays. In the presence of thymosin 4, cell migration was increased almost twofold
(69.07.1 vs. 113.35.5 cell number per field, P0.001) at 1 ng over migration in the presence of
medium containing vehicle alone (negative control). The effect of thymosin 4 on cell migration
was greatest at 1 ng/ml and at 100 and 1000 ng/ml migration was decreased. Previously, we
found that 1 ng/ml was very potent for endothelial and keratinocyte migration (13, 14).
Thymosin 4 augments the production and secretion of MMP-2 by clonogenic keratinocytes
Enzymatic degradation and remodeling of extracellular matrix, and particularly the basement
membrane that separates the epithelial (i.e., outer root sheath) and stromal (i.e., dermal sheath,
dermal papilla) compartments of the follicle, are necessary steps in normal hair development and
growth (22, 23, 25, 34). Here, we have examined the effect of thymosin 4 on the enzymatic
activity of MMPs, enzymes responsible for degradation of major extracellular matrix proteins
and, therefore, matrix and basement membrane remodeling in many biological processes,
including hair follicle development and growth (22, 25). Treatment of the clonogenic
keratinocytes with exogenous thymosin 4 caused a dose-dependent increase in the levels of
secreted and cell-derived enzyme MMP-2 (Fig. 5). MMP-2 degrades collagen type IV and

laminin, key proteins of the basement membrane. This effect on MMP-2 was specific, since the
level of another collagen IV-degrading enzyme, MMP-9, was unchanged during thymosin 4
treatment (Fig. 5). Since MMP-2 plays a role in both hair growth-associated extracellular matrix
remodeling and cell migration (2225, 3537), our data suggest that this enzyme may be a
downstream effector through which thymosin 4 exerts its effect on hair growth.
DISCUSSION
Thymosin 4 is a small 4.9-kDa molecule that functions as a major actin-sequestering protein in
cells and has many biological activities. It is upregulated during endothelial cell differentiation,
and when added exogenously, it promotes endothelial cell differentiation and migration (14, 15).
In vivo, it promotes wound repair and is a potent anti-inflammatory agent (1113, 21). Thymosin
4 is overexpressed in metastatic tumors, and its up-regulation results in increased tumor cell
motility and metastatic potential (18, 38). A related family member, thymosin 15, is also
important in the metastasis of certain tumor types (39, 40). It was reported that thymosin 4
exerts its effects on cell locomotion through specific interactions with actin that regulate
cytoskeletal organization (18, 19). Here, we show that exogenously delivered thymosin 4
promotes hair growth in normal rats and mice. When examining the distribution of endogenous
thymosin 4 through sequential phases of depilation-induced hair growth, we found that in the
resting (telogen) follicle it is expressed in the small number of cells originating in the bulge
region of the outer root sheath. As the follicles enter the active growth phase (anagen), the subset
of thymosin 4-expressing cells in the outer root sheath is expanded toward the base of the
follicle. At the peak of anagen, a significant number of thymosin 4-expressing cells are found in
the bulb area, both in the outer root sheath and among the hair matrix cells. Furthermore, isolated
clonogenic hair follicle keratinocytes, closely related, if not identical, to the hair follicle stem
cells (10, 30), produce thymosin 4 when cultured in vitro for 7-10 days. In addition, the
presence of exogenous thymosin 4 caused a dose-dependent decrease in the expression of the
stem cell marker K15 by clonogenic keratinocytes, suggesting that thymosin 4 may promote
early stem cell differentiation (i.e., transition to the TA phenotype). Most important, treatment of
the bulge-derived clonogenic keratinocytes with exogenous thymosin 4 increased their
migration and production of MMP-2.
A critical step in the hair growth cycle, at the transition between telogen and anagen, is the
movement of some of the bulge-residing stem cells downward, where their differentiated
progeny contribute to complete regrowth or regeneration of the lower, cycling portion of the
follicle (Fig. 2; refs 1, 57, 9, 10). Our data indicate that thymosin 4 facilitates this movement of
the stem cells and their immediate progeny and, thus, exerts its promoting effect on hair growth.
Apart from its direct role in cell locomotion, mediated by interaction with actin (18, 19), the
effect of thymosin 4 on MMP-2 expression appears to play an important role in this system.
MMP-2 was previously shown to contribute to cellular migration, both by means of degrading
extracellular matrix barriers for cell movement and through direct effects on cell locomotion in
vitro (37). In addition, MMP-2 is involved in hair-cycle-associated remodeling of the basement
membrane, the specialized extracellular matrix structure surrounding the epithelial core of the
follicle. Basement membrane remodeling is necessary for signaling between epithelial and
stromal elements of the growing follicle and for elongation and invasion of the lower follicle into
subcutaneous tissue during the anagen phase (2325, 35, 36).

Hair growth acceleration by thymosin 4 may also be attributed, in addition to its effects on stem
cells, to pro-angiogenic and other previously described biological activities of this molecule. It
was recently reported that VEGF promotes hair follicle development, presumably due to its
angiogenic activity (41). Thymosin 4 is angiogenic, like VEGF, and the activity of thymosin 4
may, in addition to its effects on stem cells, be due to its angiogenic activity. Recently, another
angiogenic molecule, hepatocyte growth factor, has been identified in hair follicles and found to
promote hair growth (34, 4244). Hepatocyte growth factor up-regulates thymosin 4 expression
(44) and may be acting by increasing thymosin 4 and/or synergizing with it. Furthermore,
steroids have been used successfully to treat certain types of hair loss (46). Thymosin 4 is the
anti-inflammatory molecule identified as increased in steroid-treated monocytes (21). Thus,
treatment with steroids may also involve the activity of thymosin 4 on the hair growth.
Taken together, our results suggest that thymosin 4 exerts a profound hair-promoting effect
through a combined action on several critical events in hair follicle growth, such as stem cell
progeny migration, ECM-degrading enzyme production, and differentiation. Increased
angiogenesis likely also contributes to the hair growth-promoting effects of thymosin 4.
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Received April 8, 2003; accepted October 21, 2003.

Fig. 1

Figure 1. Histological and gross appearance of skin from control and thymosin 4-treated rats and mice. For the rat
studies, animals (3-5 per group) were treated on one side of the animal with vehicle and on the other side of the same
animal with thymosin 4 and for the mouse studies (2 per group), separate mice were used for each treatment. All sections
are stained with Massons Trichrome. Microphotographs of the rat and mouse histological sections were made at x32 and
x66, respectively. a) Control vehicle-treated rat skin after 7 days. b) Control vehicle-treated mouse skin after 28 days. c)
Gross appearance of control vehicle-treated mouse skin after 28 days. d) Rat skin after 7 days of thymosin 4 treatment. e)
Mouse skin after 28 days of thymosin 4 treatment. f) Gross appearance of mice after 28 days of thymosin 4 treatment.

Fig. 2

Figure 2. Temporal and spatial distribution of thymosin 4-expressing cells during depilation-induced hair growth
cycle. C57BL/6 mouse dorsal skin of defined hair cycle stages (telogen, unmanipulated skin; early anagen, 4 days
postdepilation; late anagen, 9 days postdepilation) was harvested and processed for H&E staining (lower panels,
magnification x50) and immunohistochemical analysis of thymosin 4 expression (upper panels, magnification x320).
Telogen skin: few thymosin 4-positive-stained cells (arrowhead) are localized in the bulge region, at the site of the
insertion of arrector pili muscle. Early anagen skin: thymosin 4 expression in cells emanating from the bulge region
(arrowhead). Late anagen skin: longitudinal and transverse sections through the hair follicle basis are shown, thymosin 4expressing cells could be easily detected at the basis of the follicle, in the outer root sheath, in the portion surrounding the
matrix cells (arrowheads). Sebaceous gland (S); arrector pili muscle (AP).

Fig. 3

Figure 3. Isolation, culture, and keratin and thymosin 4 expression by clonogenic keratinocytes from rat vibrissa
bulge. a) Culture of bulge region clonogenic keratinocytes 9 days after isolation, as described in Methods. Two
clonogenic populations are shown. Magnification: x100. b) Fluorescent immunostaining of clonogenic keratinocytes with
anti-thymosin 4 antibody. Cells were incubated with primary rabbit antibody raised against full-length thymosin 4
peptide sequence, followed by staining with secondary CY3- conjugated donkey anti-rabbit antibody, as described in
Methods. Magnification x630. Control samples were processed without primary antibody, and no significant background
staining was detected (not shown). c) Pattern of keratin expression by clonogenic keratinocytes. Cells were washed with
PBS and lysed in RIPA buffer. Lysates were analyzed by Western blot with antibodies against keratin (K) 5, 6, 10, 14,
and 15, as described in Methods. Note the presence of epidermal stem cell marker K15 and absence of K10, which is an
early marker of terminal differentiation.

Fig. 4

Figure 4. Exogenous thymosin 4 decreases expression of keratin 15 in clonogenic keratinocytes. a) Western blot
analysis with anti-K15 antibody. Rat vibrissa clonogenic keratinocytes isolated from the bulge region were kept in the
serum-free medium for 3 h and then incubated for 8 h in the absence or presence of various concentrations of thymosin 4.
Cells were washed with PBS and lysed in RIPA buffer. Protein concentration was determined and lysates normalized for
equal protein were analyzed by Western blot with antibodies against keratin 15. The same membranes were reprobed with
anti-GAPDH antibody. b) Quantitation of the K15 signal was normalized to GAPDH after densitometry measurements
using NIH software.

Fig. 5

Figure 5. Effect of thymosin 4 on MMP-2 production and secretion by clonogenic keratinocytes. Cells were

incubated (16 h) in the absence or presence of increasing concentrations of thymosin 4 in serum-free medium. Cell
lysates and aliquots of the incubation media were normalized for equal cell protein and applied for zymography, as
described in Methods. Intensity of the gelatinolytic activity bands was quantitated by densitometry. a) Zymographic
analysis of secreted material (incubation medium). b and c) Quantitation of MMP-9 (b) and MMP-2 (c) gelatinolytic
activity, secreted by clonogenic keratinocytes in the absence or presence of thymosin 4. Intensity of the gelatinolytic
activity bands (quantitated by densitometry) is presented (meanSD, P=0.0368, ANOVA test).