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Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro Masahiro Kiso b, Tatsuo S. Hamazaki a, Munenari Itoh b, Sota Kikuchi b, Hidemi Nakagawa b, Hitoshi Okochi a,* a b
Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Japan Department of Dermatology, The Jikei University School of Medicine, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 December 2014 Received in revised form 31 March 2015 Accepted 17 April 2015
Background: The dermal papilla is composed of a small clump of mesenchymal cells, called dermal papilla cells (DPCs). DPCs closely interact with epidermal cells to give rise to hair follicles and shafts during hair follicle development and the hair cycle. DPCs are promising cell sources for hair regeneration therapy for alopecia patients. However, once DPCs are put into conventional two-dimensional culture conditions, they quickly lose their capability to produce hair follicles. Objective: We aimed to expand a sufficiently large population of DPCs that retain their hair inductive activity. Methods: Murine DPCs were cultured in the presence of platelet-derived growth factor-AA (PDGF-AA) and fibroblast growth factor 2 (FGF2). Expressions of follicular-related genes were analyzed by real time PCR and hair inductive activity was determined by patch assay and chamber assay in vivo. Results: FGF2 significantly increased the expression of platelet-derived growth factor receptor alpha (PDGFRa) in cultured vibrissal DPCs. PDGF-AA, a ligand of PDGFRa, promoted proliferation of DPCs synergistically when utilized with FGF2 and enhanced the expression of several follicular-related genes in DPCs. Hair reconstitution assays revealed that DPCs treated with both PDGF-AA and FGF-2 were able to maintain their hair inductive activity better than those treated with FGF2 alone. Conclusion: Both cell proliferation and hair inductive activity in murine DPCs are maintained by the synergistic effect of FGF2 and PDGF-AA. ß 2015 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
Keywords: Dermal papilla FGF2 PDGF-AA PDGFR Hair inductive activity
1. Introduction A hair follicle is an appendage of the skin, a mini organ that plays important roles on controlling body temperature and protecting delicate sites of the skin. During hair development and the hair cycle, remodeling of the hair follicle is coordinated by epithelial-mesenchymal interaction [1]. The dermal papilla (DP), which is surrounded by the dermal sheath and the hair matrix, is considered to be essential to hair induction, because implanted dermal papilla cells (DPCs) can induce hair follicles in glabrous areas such as palms and soles [2]. However, current regenerative therapy utilizing hair follicles is mainly hair replacement surgery. There are still many problems in allowing implantation of cultured
* Corresponding author at: Department of Regenerative Medicine, Research Institute, National Center for Global health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Tel.: +81 3 3202 7181. E-mail address: [email protected] (H. Okochi).
DPCs, because DPCs grow slowly and rapidly lose their hair inductive activity in vitro. We previously reported that addition of FGF2 to the medium enhances the proliferation of DPCs [3]. However, it is still difficult to obtain enough DPCs for implantation quickly and to retain the hair inductive activity of DPCs under two-dimensional culture conditions (2D). Recently, it has been reported that human dermal papilla cells have profound molecular signature changes linked to their transition from 3D to a 2D environment, with early loss of their hair inductive activity [4]. Therefore, further improvement is required for establishing suitable culture conditions for DPCs. In this study, we focused on platelet-derived growth factor (PDGF), because several reports have suggested that PDGF contributes to the induction and maintenance of anagen phase hair follicles in vivo. Comparing with wild type, postnatal PDGF-A knockout mice develop a thinner dermis, misshapen hair follicles, smaller dermal papilla, abnormal dermal sheaths, and thinner hair [5]. When PDGF-AA or PDGF-BB was injected subcutaneously into the dorsal skin of mice at telogen phase, hair follicles were
http://dx.doi.org/10.1016/j.jdermsci.2015.04.007 0923-1811/ß 2015 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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immediately induced to the anagen phase of the hair cycle [6]. Recently, it was suggested that PDGF-AA expression by immature adipocytes regulates follicular stem cell activity and that PDGFRa is activated in the DP and the lower part of hair germ during the anagen phase [7]. PDGF is a potent mitogen for cells of mesenchymal origin [8,9]. The PDGF signaling network consists of four ligands, PDGFAD, and two receptors, PDGFRa and PDGFRb, which are regarded as a receptor for tyrosine kinase (RTK) [10]. The alpha type receptor binds to PDGF-AA, PDGF-BB and PDGF-AB, whereas the beta type receptor binds with high affinity to PDGF-BB and PDGF-AB [11]. Using hair reconstitution assays, we examined the effect of PDGF on both cell proliferation of DPCs in vitro and hair inductive activity in vivo.
(Takara Bio), and ribonuclease inhibitor (Takara Bio). The primers are listed in the supplementary data (supplementary Table 1). Amplication included initial denaturation 94 8C for 3 min, 25 cycles of denaturation at 98 8C for 10 s, annealing at 62 8C for 30 s, and synthesizing 72 8C for 60 s. Quantitative PCR was carried out using a Takara SYBR Premix Ex taq (Takara Bio) according to the manufacturer’s protocol. Primers are listed in the supplementary data (supplementary Table 1). Amplication included initial denaturation at 95 8C for 10 s, 50 cycles of denaturation at 95 8C for 10 s, and annealing at 60 8C for 30 s. The expression level of the mRNA of each gene was normalized by that of OAZ-1. Scale bars were based on the standard error of mean (SEM) from three technical replicates.
2. Materials and methods
2.5. Hair reconstitution assay
2.1. Animals
The hair reconstitution assay utilized a cell mixture of cultured DPCs and fetal epidermal cells. Freshly isolated dermal and epidermal cells from C57BL/6 N mouse embryos (E17.5-18.5) were implanted as the positive controls, and only epidermal cells were implanted as the negative controls; pentobarbital sodium (1.3 mg/ kg body weight) and sevofrane were used in every experiment. For the patch assay, cell mixtures (1.0 106 epidermal cells and 2.0 106 DPCs in a volume of 50 ml PBS) were subcutaneously injected into the dorsal skin of athymic nude mice at 6 sites using an injector with a 29-gauge needle (BD, Moutain View, CA). Two weeks later, the graft sites were harvested. For the chamber assay, a silicone chamber (Taiyo Kogyo, Tokyo, Japan) was inserted subcutaneously into the dorsal midline of athymic mice. Cell mixtures (5.0 106 epidermal cells and 1.0 107 DPCs in a volume of 100 ml D-MEM) were implanted into the chamber using the pipette tips. The silicone chamber was removed one week later. Four weeks later, the graft sites were harvested. Graft skin specimens were fixed in 20% acid–alcohol– formalin overnight and then transferred to 70% ethanol. Tissues were embedded in paraffin. Then 4 mm thick serial sections were cut and stained with hematoxylin and eosin. To track donor cells from expressing GFP mice, we observed the slides with an IX70 fluorescence microscope (Olympus).
C57BL/6 mice were purchased from Clea Japan (Tokyo, Japan), and athymic nude mice (Balb/c Slc-nu/nu) were purchased from SLC Japan (Tokyo, Japan). GFP-transgenic mice were a kind gift from Dr M. Okabe, Univ. of Osaka, Japan. All the animal studies were approved by the Animal Care and Use Committee of National Center for Global Health and Medicine. 2.2. Isolation of DPCs and cell culture Vibrissal dermal papillae were obtained from 4-weeks-old C57BL/6 J mice. The mystical pad was cut open and the skin was inverted. Follicles were removed with fine forceps. The collagen capsules surrounding the vibrissae follicles were removed to expose the follicle bases, and DPs were dissected using thin needles. Isolated DPs were placed on the bottoms of cell culture dishes [3]. DPCs were cultured for 10–14 days, harvested with 0.25% trypsin–EDTA (Sigma; Shiga, Japan), and transferred to fresh culture dishes. DPCs were cultured in D-MEM low glucose supplied with 10% fetal bovine serum, with or without FGF2 (10 ng/ml: Peprotech, Rocky Hill, CA) combined with PDGF-AA (R&D Systems, Minneapolis, MN). The culture medium was exchanged every four days. Human follicle dermal papilla cells (HFDPC) were purchased from Promo Cell (Heidelberg, Germany) and were cultured in the follicle dermal papilla cell growth medium (Promo Cell) with fetal calf serum 0.04 ml/ml, bovine pituitary extract 0.004 ml/ml, FGF2 1 ng/ml, and insulin 5 mg/ml (HFDPC medium). 2.3. Immunofluorescence analysis Cultured DPCs were fixed using 4% paraformaldehyde (WAKO, Shiga, Tokyo) at room temperature for 10 min. The following primary antibodies were used: rabbit anti-PDGFRa (1:100; Cell Signaling Technology Japan; Tokyo, Japan), rabbit anti-PDGFRb (1:100; Cell Signaling Technology Japan). After primary antibodies reacted for overnight at 4 8C, they were washed out twice using 0.5% tween 20 in PBS. A secondary antibody, Alexa Fluor 594 conjugated goat anti-rabbit IgG (1:300; Invitrogen, Carlsbad, CA), was applied for 1 h at room temperature. DPCs were observed under an IX71 fluorescence microscope (Olympus). 2.4. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and statistical analysis Total RNA was prepared from DPCs using ISOGEN (WAKO) and following the manufacturer’s protocol. cDNA was synthesized from 2 mg of total RNA using PrimeScript (Takara Bio; Osaka, Japan) with a mixture of random hexamers (Takara Bio), oligo dT primers
3. Results 3.1. The expression of PDGFR in cultured DPCs To examine the effects of PDGF on DPCs, we first investigated the expression of PDGF receptor on cultured DPCs. We have previously reported that a 10 ng/ml concentration of FGF2 was suitable for DPC culture in the presence of 10% FBS [3]. We cultured the DPCs with 10% FBS [FGF2 ( )] or with 10% FBS + FGF2 (10 ng/ ml) [FGF2 (+)] for 14 days after isolation of murine vibrissal DP; then total RNA was extracted. RT-PCR was performed for 25 cycles against PDGFRa and PDGFRb, using RNA from FGF2 (+) or FGF ( ). RT-PCR analysis showed that the expression of PDGFRa was enhanced by FGF2 (Fig. 1A) and that PDGFRb was expressed in both groups. Next, quantitative PCR analysis revealed that PDGFRa expression in FGF2 (+) was about 20 times higher than that in FGF ( ). In contrast, the expression of PDGFRb in both groups was relatively low and showed no significant differences (Fig. 1B). Immunocytochemistry with P1 DPCs showed that FGF2 (+) expressed PDGFRa much more strongly than FGF2 ( ). In agreement with the PCR analysis results, PDGFRb was weakly expressed in both FGF2 (+) and FGF2 ( ) (Fig. 1C). Furthermore, we performed the same experiment using human dermal papilla cells (HFDPC). The HFDPC were disseminated at 5000/cm2 into 6 well plates and cultured
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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Fig. 1. The expression of PDGF receptors in cultured DPCs was examined with or without FGF2 (10 ng/ml): (A) RT-PCR and (B) quantitative real-time PCR were performed against PDGFRa and PDGFRb using RNA from DPCs at 14 days after primary culture. (C) Immunocytochemical staining of PDGFRa and PDGFRb in DPCs. DPCs treated with FGF2 enhanced PDGFRa but not PDGFRb expression. Scale bar = 200 mm.
with the HFDPC medium alone or addition of FGF2 (10 and 50 ng/ ml) for 4days. Both RT-PCR analysis and Immunocytochemistry showed that the expression of PDGFRa was enhanced by FGF2 (supplementary Fig. 1). These results suggested that PDGFRa expression on DPCs was enhanced by FGF2 in vitro not only in rodents but also in human.
alone was less promoted than those in FGF2 alone (Fig. 2A). We also examined cell growth of DPCs for 20 days with subculturing every 4 days (from P1 to P5). Representative data are shown in Fig. 2B. The total number of DPCs also increased with the addition of PDGF-AA in a concentration-dependent manner at all passages. This experiment was repeated three times.
3.2. Addition of PDGF together with FGF2 promote cell proliferation of DPCs in vitro
3.3. Gene analysis of DPCs by quantitative real-time PCR
Next, we examined whether cell proliferation was promoted by the addition of PDGF-AA together with FGF2. After primary culture with FGF2, DPCs were cultured under four different conditions for 5 days: (1) without FGF2 (FBS only), (2) with PDGF-AA (3) with FGF2, (4) with both FGF2 and PDGF-AA. The concentration of PDGF-AA ranged from 2 to 50 ng/ml. The total cell count of DPCs treated with both FGF2 and PDGF-AA was higher than that of DPCs treated with PDGF-AA or FGF2 alone at 3 days and was significantly increased in a concentration-dependent manner by 5 days (Fig. 2A). Cell proliferation in cultures treated with PDGF-AA
Since up-regulation of PDGFRa has been reported on DP in the anagen phase of the hair follicle [7], we used quantitative real time PCR to investigate the gene expression up-regulated at anagen phase such as versican [12,13], FGF7 [14,15], Noggin, FGF10, BMP6 [16], b-catenin [17] and ALP encoded by Akp2 [18,19] in cultured DPCs. FGF7 has been shown to participate in instructing hair germs to proliferate and initiate a new hair cycle [20]. Noggin is also regarded as a stimulator of hair-follicle induction [21,22]. Quantitative PCR was performed using RNA from both primary (P0) and passage 3 (P3) DPCs cultured for 14 days. In P0 DPCs, synergistic effect of FGF2 and PDGF-AA was observed in versican, FGF10, FGF7,
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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A
B
injected as positive controls in every experiment. As the negative control, only fetal epidermal cells were injected. Positive controls induced hair follicles (Fig. 4A), and the negative control didn’t (Fig. 4B). DPCs treated with FGF2 rarely developed hair shafts (Fig. 4C), but hair shafts were often present after FGF2 + PDGF-AA treatment (Fig. 4D). Histologically, we found mature follicles (Fig. 4E) that were sometimes associated with epidermal cysts at the upper portion (Fig. 4F), abnormal hair follicles (Fig. 4G), immature hair follicles (Fig. 4H), and epidermal cysts (Fig. 4I). Immature follicles were mainly observed after FGF2 treatment. We counted the hair follicles that developed the lower portion of the hair follicular structure, but not count abnormal hairs or immature hairs. The number of induced hair follicles arising from DPCs treated with FGF2 + PDGF-AA 20 ng/ml was greater than that of those treated with FGF2 alone at all passages (Table 1). Inductivity is defined as the percentage of successful inductions. Higher hair inductivity and more hair follicles were observed when PDGF-AA concentrations of 10–20 ng/ml were added, compared to that of the values observed with only FGF2 treatment at P3 or P5. PDGFAA at 50 ng/ml did not always result in higher inductivity and more hair follicles than that with FGF2 alone. 3.5. De novo hair follicle induction in the chamber assay
Fig. 2. (A) After DPCs were cultured with FGF2 (10 ng/ml) during primary culture, they were disseminated at 3250/cm2 into 24 well plates and cultured by without FGF2 (FBS only), with PDGF-AA alone (10–20 ng/ml) [AA], with FGF2, addition of PDGF-AA (2–50 ng/ml) together with FGF2 (FGF2 + AA) for 5 days. Total cell numbers were counted at 1, 3, or 5 days. Six independent subculture experiments were performed. The number of DPC treated with FGF2 + PDGF-AA was higher than that FGF2 alone at 3 days and by PDGF-AA in a concentration-dependent manner together with FGF2 at 5 days. (B) DPCs were disseminated at 10,000/cm2 into 6 well plates and cultured with the addition of PDGF-AA (10, 20, 50 ng/ml) for 20 days, subculturing every 4 days (from P1 to P5). Three independent subculture experiments were performed. Total cell numbers were counted at each passage. The number of DPC also increased in a concentration-dependent manner with PDGF-AA together with FGF2. Scale bars were based on the standard error of mean (SEM) from six technical replicates.
b-catenin and Akp2 gene expression while Noggin mRNA was upregulated by PDGF-AA alone and BMP6 mRNA was upregulated by FGF2 alone (Fig. 3A). On the other hand, versican and BMP6 mRNA in P3 DPCs were elevated at all concentrations of PDGF-AA compared with those in FGF2 alone. FGF7, FGF10, Noggin and bcatenin mRNA in P3 DPCs were elevated with PDGF-AA (10–20 ng/ ml) compared with those in FGF2 (Fig. 3B). Akp2 mRNA tended to be upregulated by addition of PDGF-AA in combination with FGF2 but we did not have a significant difference (Fig. 3B).
Although the number of implantations was limited to one site per mouse in the chamber assay, the hair quality and density generated with this assay is better than those in the patch assay [23]. The chamber assay was performed to compare hair inductive activity between FGF2 and FGF2 + PDGF-AA (20 ng/ml) using DPCs at P3 with fetal epidermal cells. Positive controls always induced hair follicles (Fig. 5A), and negative controls did not induce any hair follicles (Fig. 5B). Although DPCs treated with FGF2 (Fig. 5C) and DPCs treated with FGF2 and PDGF-AA (Fig. 5D) both induced hair follicles, the number of hair follicles induced by DPCs treated with FGF2 + PDGF-AA was statistically significantly higher than that with DPCs treated with FGF2 alone (Fig. 5G). Moreover, we used cultured DPCs from GFP transgenic mice to demonstrate whether the induced DP was derived from donor’s cells. GFP positive cells were observed at DP, confirming that the newly formed DPCs were derived from donor’s cells. Interestingly, we found that GFP positive cells were observed in the dermal sheath cup (DPC) (Fig. 5E).
Table 1 Follicles induced by patch assay. The number of implantation
% inductivity
Count of follicle 1
P1
P3
3.4. De novo hair follicle induction in patch assays with FGF2 or FGF2 + PDGF-AA To investigate the hair inductive ability of DPCs, both patch and chamber assays are commonly used for in vivo hair reconstitution. We first adopted the patch assay, because DPCs can be transplanted at several sites of dorsal skin of one mouse. Freshly isolated dermal and epidermal cells from mouse embryos (E17.5-18.5) were
Sample name
P5
N=9 N=9 N=9 N=6 N=6 N=6
Negative control Positive control FGF2 FGF2 + AA10 FGF2 + AA20 FGF2 + AA50
0(0/9) 100(9/9) 22(2/9) 16(1/6) 33(2/6) 16(1/6)
N=8 N=8 N=8 N=8 N=8 N=8
Negative control Positive control FGF2 FGF2 + AA10 FGF2 + AA20 FGF2 + AA50
0(0/8) 100(8/8) 37(3/8) 75(6/8) 62(5/8) 50(4/8)
2 3 2 2
N=9 N=9 N=9 N=8 N=6 N=7
Negative control Positive control FGF2 FGF2 + AA10 FGF2 + AA20 FGF2 + AA50
0(0/9) 100(9/9) 22(2/9) 33(2/8) 50(3/6) 14(1/7)
2 1 1 1
2–5
5 9
2 1 1
1 1 2
1 1
8 1 2 2
9
1
1 1
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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Fig. 3. Quantitative real-time PCR was carried out using primary and P3 DPCs cultured for 14 days. We chose genes that are especially expressed at anagen of DP to study the effect of FBS only, FBS + PDGF-AA (20 ng/ml), FBS + FGF2 and FBS + FGF2 + PDGF-AA (20 ng/ml) in primary DPCs (A) or FBS + FGF2 FBS + FGF2 + PDGF-AA (10 ng/ml) FBS + FGF2 + PDGF-AA (20 ng/ml) FBS + FGF2 + PDGF-AA (50 ng/ml) in P3 DPCs (B). The relative gene expression levels were calculated based on FBS and FGF2 group and shown as fold increase. These experiments were repeated individually three times.
4. Discussion In this study, the synergistic effect of PDGF-AA and FGF2 on cell growth of DPCs can be explained as up-regulation of PDGFRa after
treatment with FGF2. In angiogenesis, FGF2 has been reported to up-regulate PDGFRa and PDGFRb expression levels in the newly formed vessels [24]. Only PDGFRb expression has been reported in human DPCs in vitro [25], and the effect of PDGF on DPCs was not
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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Fig. 4. Mixtures of the fetal epidermal cells (E17.5-18.5) and cultured DPCs were implanted into dorsal skin subcutaneously by the injector with 29-gauge needle. (A–D) Macroscopic pictures. As the positive controls (A), freshly isolated dermal and epidermal cells from mouse embryos (E17.5-18.5) were injected, and, as the negative control (B), only epidermal cells were injected. Although DPCs treated with FGF2 did not always form a hair shaft (C), those treated with FGF2 + PDGF-AA (10–50 ng/ml) formed hair shafts (D). (E–I) Hematoxylin and eosin stain. We found mature (E) and immature follicles (F and H), abnormal hair (G), and epidermal cysts (I) in serial sections of each sample. Scale bar A–D = 1 mm, E–I = 200 mm.
considered. Recently, Festa et al. [7] reported the PDGFRa is enhanced on the DP in the anagen phase of hair follicles. We detected PDGFRb mRNA expression in murine and human DPCs, but the level of protein expression was not very strong under our culture conditions. In contrast, FGF2 dramatically increased PDGFRa expression in cultured DPCs, as shown in Fig. 1 and supplementary Fig. 1. PDGF-AA is known as a key molecule for the development of DP and the growth of hair follicles [5]. In addition, it has been suggested that PDGF-AA, but not PDGF-BB, from adipocyte precursor cells may contribute to the induction of hair follicles [7]. We assumed that PDGF-AA in combination with FGF2
might provide better conditions for culturing DPCs in vitro by maintaining the anagen phase of hair follicles. To confirm this further, we showed that the expression of the genes at the anagen phase of hair follicle were up-regulated by quantitative real time PCR. Synergistic effect of FGF2 and PDGF-AA was observed in versican, FGF10, FGF7, b-catenin and Akp2 gene expression. Among genes tested, b-catenin was reported to play important roles on the function of DP and to affect the expression of FGF7, FGF10, BMP6 [17]. On the other hand, PDGF-AA has been reported to work together with Shh to increase Noggin expression in DP [26]. Quantitative real time PCR analysis implies that PDGF-AA
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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Fig. 5. Chamber assay: mixtures of neonatal epidermal cells (E17.5-18.5) and cultured DPCs were implanted subcutaneously into silicone chamber with pipette tips. The chamber assay was performed individually five times. The results of the chamber assay showed that the positive control induced hair follicles (A), and the negative control didn’t induce any follicles (B) in every experiment, DPCs treated with FGF2 induced hair sparsely (C). DPCs treated with FGF2 + PDGF-AA (20 ng/ml) induced more hair follicles (D). The number of hair follicles induced by cells treated with FGF2 + PDGF-AA (20 ng/ml) was statistically greater than by those treated with FGF2 alone (G). When we used cultured DPCs from mice expressing GFP, GFP positive cells were detected not only at DP (white mark) but also at DSC of the formed hair follicles (red mark) (E and F). Phase contrast image (E). Fluorescent image (F). Scale bar A–D = 7 mm, E and F = 200 mm. Scale bars were based on the standard error of mean (SEM) from five technical replicates.
alone makes it possible to increase Noggin expression (Fig. 3A). These gene expressions may be closely related to maintaining hair inductive capacity during culture of DPCs. We hypothesized what might be happening in vivo from our results and summarized our findings in Fig. 6. Our patch assay showed that mature follicles were induced by addition of PDGF-AA. It is of note that, of all concentration tested, addition of PDGF-AA at 50 ng/ml showed the most proliferative effects on DPCs but the patch assay showed that, in terms of hair inductive activity, PDGF-AA (50 ng/ml) was not superior to lower concentrations. These results suggest that cell proliferation may not necessarily be correlated with hair inductive activity. In addition, we found the size of DP induced by the patch assay was
relatively large, and the hair follicle and fiber were also large and thick. It has been shown that the size of the DP is directly correlated with the original size of the hair follicle [27,28]. It has been reported that the character of induced hair follicles were retained as that of vibrissal DP [29]. In the chamber assay, GFP positive cells were observed in DPs, indicating that the hair follicles were directly induced by implanted DPCs. Moreover, we also found GFP positive cells at the dermal sheath cup (DSC). When new follicles were induced by the interaction of cultured DP with skin epithelium, newly formed dermal sheath (DS) was derived from DP [2]. On the other hand, DS division contributes directly to the recruitment of cells into the DP in early anagen, and that migration of cells into the DS causes the reduction in size of the DP during
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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Fig. 6. Schematic illustration of a possible signaling cascade in DP. The expression of PDGFRa on DPCs is enhanced by FGF2. PDGF-AA from subcutaneous adipocytes binds PDGFRa. The expression of the genes at anagen phase of hair follicle in DPCs such as FGF7, FGF10, BMP6 are up-regulated by the synergistic effect of PDGF and FGF2.
catagen [30]. In fact, cultured DSC was shown to induce hair follicles and form DP [31]. Our results suggest that implanted DPCs directly induce hair follicles and differentiate into DSC or migrate to the DSC position. Although we focused on the effect of PDGF-AA on DPCs in vitro, PDGF-AA alone stimulated growth of DPCs less than FGF2 alone (Fig. 2A). Therefore, we indicate that the synergistic effect of PDGFAA and FGF2 is important for the cultured DPCs. We previously reported that spheroid formation of cultured DPCs enhanced their hair follicle inductive ability [3]. As PDGF-AA has been shown to promote the growth of DPCs and to maintain hair follicle inductive ability in vivo [6,7], it is possible to acquire a large number of DPCs without long-term culture by employing the synergic effect of FGF2 and PDGF-AA. We anticipate that better hair follicle induction will be achieved by combining this culture method with the spheroid formation technique for DPCs. Acknowledgments We thank S. Yabe for helpful discussion, and we also thank S. Fukuda, F. Takeda and K. Nashiro for valuable technical assistance. We are grateful to Dr. Barbara Lee Smith Pierce (University of Maryland University College) for editorial work in the preparation of this manuscript. This work was supported by JSPS KAKENHI Grant Number 25670756 to H.O.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci.2015. 04.007. References [1] Schmidt-Ullrich R, Paus R. Molecular principles of hair follicle induction and morphogenesis. Bioessays 2005;27:247–61. [2] Reynolds AJ, Jahoda CA. Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis. Development 1992;115:587–93. [3] Osada A, Iwabuchi T, Kishimoto J, Hamazaki TS, Okochi H. Long-term culture of mouse vibrissal dermal papilla cells and de novo hair follicle induction. Tissue Eng 2007;13:975–82.
[4] Higgins CA, Chen JC, Cerise JE, Jahoda CA, Christiano AM. Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth. Proc Natl Acad Sci U S A 2013;110:19679–88. [5] Karlsson L, Bondjers C, Betsholtz C. Roles for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle. Development 1999;126:2611–21. [6] Tomita Y, Akiyama M, Shimizu H. PDGF isoforms induce and maintain anagen phase of murine hair follicles. J Dermatol Sci 2006;43:105–15. [7] Festa E, Fretz J, Berry R, Schmidt B, Rodeheffer M, Horowitz M, et al. Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling. Cell 2011;146:761–71. [8] Hannink M, Donoghue DJ. Structure and function of platelet-derived growth factor (PDGF) and related proteins. Biochim Biophys Acta 1989;989:1–10. [9] Heldin CH. Structural and functional studies on platelet-derived growth factor. EMBO J 1992;11:4251–9. [10] Matsui T, Heidaran M, Miki T, Popescu N, La Rochelle W, Kraus M, et al. Isolation of a novel receptor cDNA establishes the existence of two PDGF receptor genes. Science 1989;243:800–4. [11] Heidaran MA, Pierce JH, Yu JC, Lombardi D, Artrip JE, Fleming TP, et al. Role of alpha beta receptor heterodimer formation in beta platelet-derived growth factor (PDGF) receptor activation by PDGF-AB. J Biol Chem 1991;266:20232– 37. [12] du Cros DL, LeBaron RG, Couchman JR. Association of versican with dermal matrices and its potential role in hair follicle development and cycling. J Invest Dermatol 1995;105:426–31. [13] Kishimoto J, Ehama R, Wu L, Jiang S, Jiang N, Burgeson RE. Selective activation of the versican promoter by epithelial- mesenchymal interactions during hair follicle development. Proc Natl Acad Sci U S A 1999;96:7336–41. [14] Guo L, Degenstein L, Fuchs E. Keratinocyte growth factor is required for hair development but not for wound healing. Genes Dev 1996;10:165–75. [15] Rosenquist TA, Martin GR. Fibroblast growth factor signalling in the hair growth cycle: expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle. Dev Dyn 1996;205:379–86. [16] Rendl M, Polak L, Fuchs E. BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties. Genes Dev 2008;22: 543–57. [17] Enshell-Seijffers D, Lindon C, Kashiwagi M. Morgan BA: beta-catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair. Dev Cell 2010;18:633–42. [18] Handjiski BK, Eichmuller S, Hofmann U, Czarnetzki BM, Paus R. Alkaline phosphatase activity and localization during the murine hair cycle. Br J Dermatol 1994;131:303–10. [19] Rendl M, Lewis L, Fuchs E. Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol 2005;3:e331. [20] Greco V, Chen T, Rendl M, Schober M, Pasolli HA, Stokes N, et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 2009;4:155–69. [21] Botchkarev VA, Botchkareva NV, Roth W, Nakamura M, Chen LH, Herzog W, et al. Noggin is a mesenchymally derived stimulator of hair-follicle induction. Nat Cell Biol 1999;1:158–64. [22] Jamora C, DasGupta R, Kocieniewski P, Fuchs E. Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 2003;422:317–22. [23] Liang Y, Silva KA, Kennedy V, Sundberg JP. Comparisons of mouse models for hair follicle reconstitution. Exp Dermatol 2011;20:1011–5.
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[28] Ibrahim L, Wright EA. A quantitative study of hair growth using mouse and rat vibrissal follicles, I. Dermal papilla volume determines hair volume. J Embryol Exp Morphol 1982;72:209–24. [29] Jahoda CA, Reynolds AJ, Oliver RF. Induction of hair growth in ear wounds by cultured dermal papilla cells. J Invest Dermatol 1993;101:584–90. [30] Tobin DJ, Gunin A, Magerl M, Handijski B, Paus R. Plasticity and cytokinetic dynamics of the hair follicle mesenchyme: implications for hair growth control. J Invest Dermatol 2003;120:895–904. [31] McElwee KJ, Kissling S, Wenzel E, Huth A, Hoffmann R. Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla. J Invest Dermatol 2003;121: 1267–75.
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Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro Masahiro Kiso b, Tatsuo S. Hamazaki a, Munenari Itoh b, Sota Kikuchi b, Hidemi Nakagawa b, Hitoshi Okochi a,* a b
Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Japan Department of Dermatology, The Jikei University School of Medicine, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 December 2014 Received in revised form 31 March 2015 Accepted 17 April 2015
Background: The dermal papilla is composed of a small clump of mesenchymal cells, called dermal papilla cells (DPCs). DPCs closely interact with epidermal cells to give rise to hair follicles and shafts during hair follicle development and the hair cycle. DPCs are promising cell sources for hair regeneration therapy for alopecia patients. However, once DPCs are put into conventional two-dimensional culture conditions, they quickly lose their capability to produce hair follicles. Objective: We aimed to expand a sufficiently large population of DPCs that retain their hair inductive activity. Methods: Murine DPCs were cultured in the presence of platelet-derived growth factor-AA (PDGF-AA) and fibroblast growth factor 2 (FGF2). Expressions of follicular-related genes were analyzed by real time PCR and hair inductive activity was determined by patch assay and chamber assay in vivo. Results: FGF2 significantly increased the expression of platelet-derived growth factor receptor alpha (PDGFRa) in cultured vibrissal DPCs. PDGF-AA, a ligand of PDGFRa, promoted proliferation of DPCs synergistically when utilized with FGF2 and enhanced the expression of several follicular-related genes in DPCs. Hair reconstitution assays revealed that DPCs treated with both PDGF-AA and FGF-2 were able to maintain their hair inductive activity better than those treated with FGF2 alone. Conclusion: Both cell proliferation and hair inductive activity in murine DPCs are maintained by the synergistic effect of FGF2 and PDGF-AA. ß 2015 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
Keywords: Dermal papilla FGF2 PDGF-AA PDGFR Hair inductive activity
1. Introduction A hair follicle is an appendage of the skin, a mini organ that plays important roles on controlling body temperature and protecting delicate sites of the skin. During hair development and the hair cycle, remodeling of the hair follicle is coordinated by epithelial-mesenchymal interaction [1]. The dermal papilla (DP), which is surrounded by the dermal sheath and the hair matrix, is considered to be essential to hair induction, because implanted dermal papilla cells (DPCs) can induce hair follicles in glabrous areas such as palms and soles [2]. However, current regenerative therapy utilizing hair follicles is mainly hair replacement surgery. There are still many problems in allowing implantation of cultured
* Corresponding author at: Department of Regenerative Medicine, Research Institute, National Center for Global health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Tel.: +81 3 3202 7181. E-mail address: [email protected] (H. Okochi).
DPCs, because DPCs grow slowly and rapidly lose their hair inductive activity in vitro. We previously reported that addition of FGF2 to the medium enhances the proliferation of DPCs [3]. However, it is still difficult to obtain enough DPCs for implantation quickly and to retain the hair inductive activity of DPCs under two-dimensional culture conditions (2D). Recently, it has been reported that human dermal papilla cells have profound molecular signature changes linked to their transition from 3D to a 2D environment, with early loss of their hair inductive activity [4]. Therefore, further improvement is required for establishing suitable culture conditions for DPCs. In this study, we focused on platelet-derived growth factor (PDGF), because several reports have suggested that PDGF contributes to the induction and maintenance of anagen phase hair follicles in vivo. Comparing with wild type, postnatal PDGF-A knockout mice develop a thinner dermis, misshapen hair follicles, smaller dermal papilla, abnormal dermal sheaths, and thinner hair [5]. When PDGF-AA or PDGF-BB was injected subcutaneously into the dorsal skin of mice at telogen phase, hair follicles were
http://dx.doi.org/10.1016/j.jdermsci.2015.04.007 0923-1811/ß 2015 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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immediately induced to the anagen phase of the hair cycle [6]. Recently, it was suggested that PDGF-AA expression by immature adipocytes regulates follicular stem cell activity and that PDGFRa is activated in the DP and the lower part of hair germ during the anagen phase [7]. PDGF is a potent mitogen for cells of mesenchymal origin [8,9]. The PDGF signaling network consists of four ligands, PDGFAD, and two receptors, PDGFRa and PDGFRb, which are regarded as a receptor for tyrosine kinase (RTK) [10]. The alpha type receptor binds to PDGF-AA, PDGF-BB and PDGF-AB, whereas the beta type receptor binds with high affinity to PDGF-BB and PDGF-AB [11]. Using hair reconstitution assays, we examined the effect of PDGF on both cell proliferation of DPCs in vitro and hair inductive activity in vivo.
(Takara Bio), and ribonuclease inhibitor (Takara Bio). The primers are listed in the supplementary data (supplementary Table 1). Amplication included initial denaturation 94 8C for 3 min, 25 cycles of denaturation at 98 8C for 10 s, annealing at 62 8C for 30 s, and synthesizing 72 8C for 60 s. Quantitative PCR was carried out using a Takara SYBR Premix Ex taq (Takara Bio) according to the manufacturer’s protocol. Primers are listed in the supplementary data (supplementary Table 1). Amplication included initial denaturation at 95 8C for 10 s, 50 cycles of denaturation at 95 8C for 10 s, and annealing at 60 8C for 30 s. The expression level of the mRNA of each gene was normalized by that of OAZ-1. Scale bars were based on the standard error of mean (SEM) from three technical replicates.
2. Materials and methods
2.5. Hair reconstitution assay
2.1. Animals
The hair reconstitution assay utilized a cell mixture of cultured DPCs and fetal epidermal cells. Freshly isolated dermal and epidermal cells from C57BL/6 N mouse embryos (E17.5-18.5) were implanted as the positive controls, and only epidermal cells were implanted as the negative controls; pentobarbital sodium (1.3 mg/ kg body weight) and sevofrane were used in every experiment. For the patch assay, cell mixtures (1.0 106 epidermal cells and 2.0 106 DPCs in a volume of 50 ml PBS) were subcutaneously injected into the dorsal skin of athymic nude mice at 6 sites using an injector with a 29-gauge needle (BD, Moutain View, CA). Two weeks later, the graft sites were harvested. For the chamber assay, a silicone chamber (Taiyo Kogyo, Tokyo, Japan) was inserted subcutaneously into the dorsal midline of athymic mice. Cell mixtures (5.0 106 epidermal cells and 1.0 107 DPCs in a volume of 100 ml D-MEM) were implanted into the chamber using the pipette tips. The silicone chamber was removed one week later. Four weeks later, the graft sites were harvested. Graft skin specimens were fixed in 20% acid–alcohol– formalin overnight and then transferred to 70% ethanol. Tissues were embedded in paraffin. Then 4 mm thick serial sections were cut and stained with hematoxylin and eosin. To track donor cells from expressing GFP mice, we observed the slides with an IX70 fluorescence microscope (Olympus).
C57BL/6 mice were purchased from Clea Japan (Tokyo, Japan), and athymic nude mice (Balb/c Slc-nu/nu) were purchased from SLC Japan (Tokyo, Japan). GFP-transgenic mice were a kind gift from Dr M. Okabe, Univ. of Osaka, Japan. All the animal studies were approved by the Animal Care and Use Committee of National Center for Global Health and Medicine. 2.2. Isolation of DPCs and cell culture Vibrissal dermal papillae were obtained from 4-weeks-old C57BL/6 J mice. The mystical pad was cut open and the skin was inverted. Follicles were removed with fine forceps. The collagen capsules surrounding the vibrissae follicles were removed to expose the follicle bases, and DPs were dissected using thin needles. Isolated DPs were placed on the bottoms of cell culture dishes [3]. DPCs were cultured for 10–14 days, harvested with 0.25% trypsin–EDTA (Sigma; Shiga, Japan), and transferred to fresh culture dishes. DPCs were cultured in D-MEM low glucose supplied with 10% fetal bovine serum, with or without FGF2 (10 ng/ml: Peprotech, Rocky Hill, CA) combined with PDGF-AA (R&D Systems, Minneapolis, MN). The culture medium was exchanged every four days. Human follicle dermal papilla cells (HFDPC) were purchased from Promo Cell (Heidelberg, Germany) and were cultured in the follicle dermal papilla cell growth medium (Promo Cell) with fetal calf serum 0.04 ml/ml, bovine pituitary extract 0.004 ml/ml, FGF2 1 ng/ml, and insulin 5 mg/ml (HFDPC medium). 2.3. Immunofluorescence analysis Cultured DPCs were fixed using 4% paraformaldehyde (WAKO, Shiga, Tokyo) at room temperature for 10 min. The following primary antibodies were used: rabbit anti-PDGFRa (1:100; Cell Signaling Technology Japan; Tokyo, Japan), rabbit anti-PDGFRb (1:100; Cell Signaling Technology Japan). After primary antibodies reacted for overnight at 4 8C, they were washed out twice using 0.5% tween 20 in PBS. A secondary antibody, Alexa Fluor 594 conjugated goat anti-rabbit IgG (1:300; Invitrogen, Carlsbad, CA), was applied for 1 h at room temperature. DPCs were observed under an IX71 fluorescence microscope (Olympus). 2.4. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and statistical analysis Total RNA was prepared from DPCs using ISOGEN (WAKO) and following the manufacturer’s protocol. cDNA was synthesized from 2 mg of total RNA using PrimeScript (Takara Bio; Osaka, Japan) with a mixture of random hexamers (Takara Bio), oligo dT primers
3. Results 3.1. The expression of PDGFR in cultured DPCs To examine the effects of PDGF on DPCs, we first investigated the expression of PDGF receptor on cultured DPCs. We have previously reported that a 10 ng/ml concentration of FGF2 was suitable for DPC culture in the presence of 10% FBS [3]. We cultured the DPCs with 10% FBS [FGF2 ( )] or with 10% FBS + FGF2 (10 ng/ ml) [FGF2 (+)] for 14 days after isolation of murine vibrissal DP; then total RNA was extracted. RT-PCR was performed for 25 cycles against PDGFRa and PDGFRb, using RNA from FGF2 (+) or FGF ( ). RT-PCR analysis showed that the expression of PDGFRa was enhanced by FGF2 (Fig. 1A) and that PDGFRb was expressed in both groups. Next, quantitative PCR analysis revealed that PDGFRa expression in FGF2 (+) was about 20 times higher than that in FGF ( ). In contrast, the expression of PDGFRb in both groups was relatively low and showed no significant differences (Fig. 1B). Immunocytochemistry with P1 DPCs showed that FGF2 (+) expressed PDGFRa much more strongly than FGF2 ( ). In agreement with the PCR analysis results, PDGFRb was weakly expressed in both FGF2 (+) and FGF2 ( ) (Fig. 1C). Furthermore, we performed the same experiment using human dermal papilla cells (HFDPC). The HFDPC were disseminated at 5000/cm2 into 6 well plates and cultured
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Fig. 1. The expression of PDGF receptors in cultured DPCs was examined with or without FGF2 (10 ng/ml): (A) RT-PCR and (B) quantitative real-time PCR were performed against PDGFRa and PDGFRb using RNA from DPCs at 14 days after primary culture. (C) Immunocytochemical staining of PDGFRa and PDGFRb in DPCs. DPCs treated with FGF2 enhanced PDGFRa but not PDGFRb expression. Scale bar = 200 mm.
with the HFDPC medium alone or addition of FGF2 (10 and 50 ng/ ml) for 4days. Both RT-PCR analysis and Immunocytochemistry showed that the expression of PDGFRa was enhanced by FGF2 (supplementary Fig. 1). These results suggested that PDGFRa expression on DPCs was enhanced by FGF2 in vitro not only in rodents but also in human.
alone was less promoted than those in FGF2 alone (Fig. 2A). We also examined cell growth of DPCs for 20 days with subculturing every 4 days (from P1 to P5). Representative data are shown in Fig. 2B. The total number of DPCs also increased with the addition of PDGF-AA in a concentration-dependent manner at all passages. This experiment was repeated three times.
3.2. Addition of PDGF together with FGF2 promote cell proliferation of DPCs in vitro
3.3. Gene analysis of DPCs by quantitative real-time PCR
Next, we examined whether cell proliferation was promoted by the addition of PDGF-AA together with FGF2. After primary culture with FGF2, DPCs were cultured under four different conditions for 5 days: (1) without FGF2 (FBS only), (2) with PDGF-AA (3) with FGF2, (4) with both FGF2 and PDGF-AA. The concentration of PDGF-AA ranged from 2 to 50 ng/ml. The total cell count of DPCs treated with both FGF2 and PDGF-AA was higher than that of DPCs treated with PDGF-AA or FGF2 alone at 3 days and was significantly increased in a concentration-dependent manner by 5 days (Fig. 2A). Cell proliferation in cultures treated with PDGF-AA
Since up-regulation of PDGFRa has been reported on DP in the anagen phase of the hair follicle [7], we used quantitative real time PCR to investigate the gene expression up-regulated at anagen phase such as versican [12,13], FGF7 [14,15], Noggin, FGF10, BMP6 [16], b-catenin [17] and ALP encoded by Akp2 [18,19] in cultured DPCs. FGF7 has been shown to participate in instructing hair germs to proliferate and initiate a new hair cycle [20]. Noggin is also regarded as a stimulator of hair-follicle induction [21,22]. Quantitative PCR was performed using RNA from both primary (P0) and passage 3 (P3) DPCs cultured for 14 days. In P0 DPCs, synergistic effect of FGF2 and PDGF-AA was observed in versican, FGF10, FGF7,
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B
injected as positive controls in every experiment. As the negative control, only fetal epidermal cells were injected. Positive controls induced hair follicles (Fig. 4A), and the negative control didn’t (Fig. 4B). DPCs treated with FGF2 rarely developed hair shafts (Fig. 4C), but hair shafts were often present after FGF2 + PDGF-AA treatment (Fig. 4D). Histologically, we found mature follicles (Fig. 4E) that were sometimes associated with epidermal cysts at the upper portion (Fig. 4F), abnormal hair follicles (Fig. 4G), immature hair follicles (Fig. 4H), and epidermal cysts (Fig. 4I). Immature follicles were mainly observed after FGF2 treatment. We counted the hair follicles that developed the lower portion of the hair follicular structure, but not count abnormal hairs or immature hairs. The number of induced hair follicles arising from DPCs treated with FGF2 + PDGF-AA 20 ng/ml was greater than that of those treated with FGF2 alone at all passages (Table 1). Inductivity is defined as the percentage of successful inductions. Higher hair inductivity and more hair follicles were observed when PDGF-AA concentrations of 10–20 ng/ml were added, compared to that of the values observed with only FGF2 treatment at P3 or P5. PDGFAA at 50 ng/ml did not always result in higher inductivity and more hair follicles than that with FGF2 alone. 3.5. De novo hair follicle induction in the chamber assay
Fig. 2. (A) After DPCs were cultured with FGF2 (10 ng/ml) during primary culture, they were disseminated at 3250/cm2 into 24 well plates and cultured by without FGF2 (FBS only), with PDGF-AA alone (10–20 ng/ml) [AA], with FGF2, addition of PDGF-AA (2–50 ng/ml) together with FGF2 (FGF2 + AA) for 5 days. Total cell numbers were counted at 1, 3, or 5 days. Six independent subculture experiments were performed. The number of DPC treated with FGF2 + PDGF-AA was higher than that FGF2 alone at 3 days and by PDGF-AA in a concentration-dependent manner together with FGF2 at 5 days. (B) DPCs were disseminated at 10,000/cm2 into 6 well plates and cultured with the addition of PDGF-AA (10, 20, 50 ng/ml) for 20 days, subculturing every 4 days (from P1 to P5). Three independent subculture experiments were performed. Total cell numbers were counted at each passage. The number of DPC also increased in a concentration-dependent manner with PDGF-AA together with FGF2. Scale bars were based on the standard error of mean (SEM) from six technical replicates.
b-catenin and Akp2 gene expression while Noggin mRNA was upregulated by PDGF-AA alone and BMP6 mRNA was upregulated by FGF2 alone (Fig. 3A). On the other hand, versican and BMP6 mRNA in P3 DPCs were elevated at all concentrations of PDGF-AA compared with those in FGF2 alone. FGF7, FGF10, Noggin and bcatenin mRNA in P3 DPCs were elevated with PDGF-AA (10–20 ng/ ml) compared with those in FGF2 (Fig. 3B). Akp2 mRNA tended to be upregulated by addition of PDGF-AA in combination with FGF2 but we did not have a significant difference (Fig. 3B).
Although the number of implantations was limited to one site per mouse in the chamber assay, the hair quality and density generated with this assay is better than those in the patch assay [23]. The chamber assay was performed to compare hair inductive activity between FGF2 and FGF2 + PDGF-AA (20 ng/ml) using DPCs at P3 with fetal epidermal cells. Positive controls always induced hair follicles (Fig. 5A), and negative controls did not induce any hair follicles (Fig. 5B). Although DPCs treated with FGF2 (Fig. 5C) and DPCs treated with FGF2 and PDGF-AA (Fig. 5D) both induced hair follicles, the number of hair follicles induced by DPCs treated with FGF2 + PDGF-AA was statistically significantly higher than that with DPCs treated with FGF2 alone (Fig. 5G). Moreover, we used cultured DPCs from GFP transgenic mice to demonstrate whether the induced DP was derived from donor’s cells. GFP positive cells were observed at DP, confirming that the newly formed DPCs were derived from donor’s cells. Interestingly, we found that GFP positive cells were observed in the dermal sheath cup (DPC) (Fig. 5E).
Table 1 Follicles induced by patch assay. The number of implantation
% inductivity
Count of follicle 1
P1
P3
3.4. De novo hair follicle induction in patch assays with FGF2 or FGF2 + PDGF-AA To investigate the hair inductive ability of DPCs, both patch and chamber assays are commonly used for in vivo hair reconstitution. We first adopted the patch assay, because DPCs can be transplanted at several sites of dorsal skin of one mouse. Freshly isolated dermal and epidermal cells from mouse embryos (E17.5-18.5) were
Sample name
P5
N=9 N=9 N=9 N=6 N=6 N=6
Negative control Positive control FGF2 FGF2 + AA10 FGF2 + AA20 FGF2 + AA50
0(0/9) 100(9/9) 22(2/9) 16(1/6) 33(2/6) 16(1/6)
N=8 N=8 N=8 N=8 N=8 N=8
Negative control Positive control FGF2 FGF2 + AA10 FGF2 + AA20 FGF2 + AA50
0(0/8) 100(8/8) 37(3/8) 75(6/8) 62(5/8) 50(4/8)
2 3 2 2
N=9 N=9 N=9 N=8 N=6 N=7
Negative control Positive control FGF2 FGF2 + AA10 FGF2 + AA20 FGF2 + AA50
0(0/9) 100(9/9) 22(2/9) 33(2/8) 50(3/6) 14(1/7)
2 1 1 1
2–5
5 9
2 1 1
1 1 2
1 1
8 1 2 2
9
1
1 1
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Fig. 3. Quantitative real-time PCR was carried out using primary and P3 DPCs cultured for 14 days. We chose genes that are especially expressed at anagen of DP to study the effect of FBS only, FBS + PDGF-AA (20 ng/ml), FBS + FGF2 and FBS + FGF2 + PDGF-AA (20 ng/ml) in primary DPCs (A) or FBS + FGF2 FBS + FGF2 + PDGF-AA (10 ng/ml) FBS + FGF2 + PDGF-AA (20 ng/ml) FBS + FGF2 + PDGF-AA (50 ng/ml) in P3 DPCs (B). The relative gene expression levels were calculated based on FBS and FGF2 group and shown as fold increase. These experiments were repeated individually three times.
4. Discussion In this study, the synergistic effect of PDGF-AA and FGF2 on cell growth of DPCs can be explained as up-regulation of PDGFRa after
treatment with FGF2. In angiogenesis, FGF2 has been reported to up-regulate PDGFRa and PDGFRb expression levels in the newly formed vessels [24]. Only PDGFRb expression has been reported in human DPCs in vitro [25], and the effect of PDGF on DPCs was not
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Fig. 4. Mixtures of the fetal epidermal cells (E17.5-18.5) and cultured DPCs were implanted into dorsal skin subcutaneously by the injector with 29-gauge needle. (A–D) Macroscopic pictures. As the positive controls (A), freshly isolated dermal and epidermal cells from mouse embryos (E17.5-18.5) were injected, and, as the negative control (B), only epidermal cells were injected. Although DPCs treated with FGF2 did not always form a hair shaft (C), those treated with FGF2 + PDGF-AA (10–50 ng/ml) formed hair shafts (D). (E–I) Hematoxylin and eosin stain. We found mature (E) and immature follicles (F and H), abnormal hair (G), and epidermal cysts (I) in serial sections of each sample. Scale bar A–D = 1 mm, E–I = 200 mm.
considered. Recently, Festa et al. [7] reported the PDGFRa is enhanced on the DP in the anagen phase of hair follicles. We detected PDGFRb mRNA expression in murine and human DPCs, but the level of protein expression was not very strong under our culture conditions. In contrast, FGF2 dramatically increased PDGFRa expression in cultured DPCs, as shown in Fig. 1 and supplementary Fig. 1. PDGF-AA is known as a key molecule for the development of DP and the growth of hair follicles [5]. In addition, it has been suggested that PDGF-AA, but not PDGF-BB, from adipocyte precursor cells may contribute to the induction of hair follicles [7]. We assumed that PDGF-AA in combination with FGF2
might provide better conditions for culturing DPCs in vitro by maintaining the anagen phase of hair follicles. To confirm this further, we showed that the expression of the genes at the anagen phase of hair follicle were up-regulated by quantitative real time PCR. Synergistic effect of FGF2 and PDGF-AA was observed in versican, FGF10, FGF7, b-catenin and Akp2 gene expression. Among genes tested, b-catenin was reported to play important roles on the function of DP and to affect the expression of FGF7, FGF10, BMP6 [17]. On the other hand, PDGF-AA has been reported to work together with Shh to increase Noggin expression in DP [26]. Quantitative real time PCR analysis implies that PDGF-AA
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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Fig. 5. Chamber assay: mixtures of neonatal epidermal cells (E17.5-18.5) and cultured DPCs were implanted subcutaneously into silicone chamber with pipette tips. The chamber assay was performed individually five times. The results of the chamber assay showed that the positive control induced hair follicles (A), and the negative control didn’t induce any follicles (B) in every experiment, DPCs treated with FGF2 induced hair sparsely (C). DPCs treated with FGF2 + PDGF-AA (20 ng/ml) induced more hair follicles (D). The number of hair follicles induced by cells treated with FGF2 + PDGF-AA (20 ng/ml) was statistically greater than by those treated with FGF2 alone (G). When we used cultured DPCs from mice expressing GFP, GFP positive cells were detected not only at DP (white mark) but also at DSC of the formed hair follicles (red mark) (E and F). Phase contrast image (E). Fluorescent image (F). Scale bar A–D = 7 mm, E and F = 200 mm. Scale bars were based on the standard error of mean (SEM) from five technical replicates.
alone makes it possible to increase Noggin expression (Fig. 3A). These gene expressions may be closely related to maintaining hair inductive capacity during culture of DPCs. We hypothesized what might be happening in vivo from our results and summarized our findings in Fig. 6. Our patch assay showed that mature follicles were induced by addition of PDGF-AA. It is of note that, of all concentration tested, addition of PDGF-AA at 50 ng/ml showed the most proliferative effects on DPCs but the patch assay showed that, in terms of hair inductive activity, PDGF-AA (50 ng/ml) was not superior to lower concentrations. These results suggest that cell proliferation may not necessarily be correlated with hair inductive activity. In addition, we found the size of DP induced by the patch assay was
relatively large, and the hair follicle and fiber were also large and thick. It has been shown that the size of the DP is directly correlated with the original size of the hair follicle [27,28]. It has been reported that the character of induced hair follicles were retained as that of vibrissal DP [29]. In the chamber assay, GFP positive cells were observed in DPs, indicating that the hair follicles were directly induced by implanted DPCs. Moreover, we also found GFP positive cells at the dermal sheath cup (DSC). When new follicles were induced by the interaction of cultured DP with skin epithelium, newly formed dermal sheath (DS) was derived from DP [2]. On the other hand, DS division contributes directly to the recruitment of cells into the DP in early anagen, and that migration of cells into the DS causes the reduction in size of the DP during
Please cite this article in press as: Kiso M, et al. Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.007
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Fig. 6. Schematic illustration of a possible signaling cascade in DP. The expression of PDGFRa on DPCs is enhanced by FGF2. PDGF-AA from subcutaneous adipocytes binds PDGFRa. The expression of the genes at anagen phase of hair follicle in DPCs such as FGF7, FGF10, BMP6 are up-regulated by the synergistic effect of PDGF and FGF2.
catagen [30]. In fact, cultured DSC was shown to induce hair follicles and form DP [31]. Our results suggest that implanted DPCs directly induce hair follicles and differentiate into DSC or migrate to the DSC position. Although we focused on the effect of PDGF-AA on DPCs in vitro, PDGF-AA alone stimulated growth of DPCs less than FGF2 alone (Fig. 2A). Therefore, we indicate that the synergistic effect of PDGFAA and FGF2 is important for the cultured DPCs. We previously reported that spheroid formation of cultured DPCs enhanced their hair follicle inductive ability [3]. As PDGF-AA has been shown to promote the growth of DPCs and to maintain hair follicle inductive ability in vivo [6,7], it is possible to acquire a large number of DPCs without long-term culture by employing the synergic effect of FGF2 and PDGF-AA. We anticipate that better hair follicle induction will be achieved by combining this culture method with the spheroid formation technique for DPCs. Acknowledgments We thank S. Yabe for helpful discussion, and we also thank S. Fukuda, F. Takeda and K. Nashiro for valuable technical assistance. We are grateful to Dr. Barbara Lee Smith Pierce (University of Maryland University College) for editorial work in the preparation of this manuscript. This work was supported by JSPS KAKENHI Grant Number 25670756 to H.O.
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