- Email: [email protected]
Bulge cells of human hair follicles: segregation, cultivation and properties
Colloids and Surfaces B: Biointerfaces 47 (2006) 50–56
Bulge cells of human hair follicles: segregation, cultivation and properties Yi Zhang b,1 , Mingming Xiang a , Yun Wang b , Jun Yan b , Yijun Zeng b , Jin Yu b , Tian Yang b,∗ a
Department of Dermatology of Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China b Department of Cell Biology, Third Military Medical University, Chongqing 400038, PR China Received 19 April 2005; received in revised form 12 October 2005; accepted 21 November 2005 Available online 4 January 2006
Abstract The bulge region of hair follicle has been reported as a putative reservoir of hair follicle stem cell (HFSC) for years; however, few studies were done about the characteristics of bulge-originated cells in vitro up to now. In this experiment, the bulge cells isolated from human hair follicles by enzymatic digestion and microdissection were cultured and passaged, and the morphological and biological features of cultured bulge cells were investigated by microscopy and immunocytochemistry. The result showed that new-proliferated cells could be observed on the second day after inoculation, and the quantity of the cells with a greater proliferation potential, reached a peak at the 6th day and maintained this higher level for several days. The mitotic figures of bulge cells were seen and these cells showed undifferentiated morphologic features. The bulge cells strongly expressed K19 and 1-integrin, which are the markers of HFSC, in a descensive way with the culture time. The result indicates that the cultured bulge cell from human hair follicle possesses the properties of primitive cells and supports the hypothesis that HFSC resides in the bulge area. © 2005 Elsevier B.V. All rights reserved. Keywords: Hair follicle; Bulge; Stem cell
1. Introduction The epidermis is the outermost layer of the skin and contacts the external environment directly. It is in continuity with epidermal appendages, such as hair follicle, sebaceous gland and sweat gland, which form through complex epithelio-mesenchymal interactions during embryonic life. Unique to the hair follicle, the epidermis contains a single type of keratinocytes, while hair follicle consists of several different specialized epithelial and connective tissue layers, surrounded by a vascular bed and a neural network. These include three cell types of the hair shaft (medulla, cortex and cuticle), three of inner root sheath (Henle’s and Huxley’s layer, the cuticle) and the outer root sheath. Hair follicle undergoes dynamic changes from an active growing phase (anagen), to a remodeling phase (catagen), and finally to a quiescent phase (telogen). Early in 1954, Chase postulated that the “upper outer root sheath” contains a population of multipotential stem cells capable of not only forming the follicle, but the epidermis and the sebaceous gland. How-
∗ 1
Corresponding author. Tel.: +86 23 68752260; fax: +86 23 65463056. E-mail addresses: [email protected] (Y. Zhang), [email protected] (T. Yang). Tel.: +86 23 68752259.
0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2005.11.017
ever, a major obstacle in this field had been lacking of operative segregation methods and reliable molecular markers. Epidermal appendages have long been known to contribute to hair regeneration and cycle maintenance [1]. It is found that adult stem cells were slow-cycling and self-renewal in vivo, and responsible for the long-term maintenance of the tissues. Many investigators made use of cell kinetics to distinguish the stem cells from the more transit amplifying (TA) cells [2–4]. Al-Barwari and Potten, using [3 H] thymidine to evaluate label retention in murine haired epidermis, discovered that the majority of label-retaining cells (LRCs) in the skin were restricted to the bulge region of the hair follicles [26]. As the bulge represents a slow-cycling population [2,5,6], and may regenerate complete hair follicle after X-irradiation or after removal of the lower parts of the hair follicles [7], it has been proposed that these cells represent multipotent stem cells in the skin, termed as hair follicle stem cell (HFSC). Although more than 10 years has passed since hair follicle bulge was thought to contain stem cells of interfollicular epithelium, the characteristic and the behavior of hair follicle stem cells outside the niche have not been fully understood yet. An extensive knowledge about the bulge is crucial to answer this question. A recent observation in the field of skin biology was that epidermal keratinocytes could be grown in culture and the
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 47 (2006) 50–56
properties of the cells were also discussed [8,9]. Recent years, many investigators were devoted to establish culture of hair follicle cells of mouse, rat and human [10–12]. Growth of human hair keratinocytes in primary culture has been reported using explant culture of plucked hair follicles, but the exact characteristic of cells from bulge region has not been examined in detail. In this report, we described a modified method to harvest intact hair follicles. The bulge, microdissected on the basis of morphologic feature, were cultured and passaged successfully, and then the biologic nature and the expression of markers of bulge cells were also studied. We believe that the work on the characteristic of hair follicle stem cells is essential for understanding follicle regeneration and cycle maintenance. 2. Materials and methods
51
2.4. Subculture and amplification After 2 weeks in primary culture, cells were collected by incubation with a 1:1 mixture of 0.125% trypsin (Sigma) and 0.02% EDTA (Sigma) for 15 min at 37 ◦ C. The dispersed cells were centrifuged for 10 min at 200 × g and replanted in 50 cm2 tissue culture flasks with a medium change every 3–4 days. Cells were routinely passaged every 5–7 days. For the sake of preservation, the cells were digested from culture dishes as described above. After one rinse with culture medium, the cells were resuspended in 1 ml fetal calf serum containing 10% dimethyl sulfoxide (Sigma), transferred into a cryotube, and placed into a freezer in a 4, −20, and −80 ◦ C container by turns. After 24 h the cryotubes were transferred into a liquid nitrogen tank.
2.1. Media 2.5. Determination of growth curve Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (3:1) (Hyclone), supplemented with 10% fetal bovine serum (Hyclone), 10 ng/ml epidermal growth factor (Sigma), hydrocortisone, 5 g/ml insulin (Sigma) and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin). William’E medium (Sigma), also supplemented with 10% fetal bovine serum, 10 ng/ml epidermal growth factor, hydrocortisone, 5 g/ml insulin and antibiotics. Serum-free DMEM/F12 (3:1) was used in some experiments. To examine the effects of various medium conditions on cell growth, we measured the diameter of the cell sheets.
Cells were planted into 4 × 6-well plates at a density of 1 × 104 cells/well. After 2 days in culture, three wells were trypsinized and counted at day 1, 2, 3, 4, 5, 6, 7, 8 and 9 days, respectively. The growth curve was protracted from the mean cell number at each time point. 2.6. Immunohistochemical staining
Plastic surgery specimens of human scalp skin were obtained from the nape of the neck. After rinsing with 70% alcohol, the tissues were trimmed into small pieces (10 mm × 4 mm), and the skin fragments were incubated in 0.25% dispase II for 12 h at 4 ◦ C. The hair follicles were squeezed out carefully. Those in anagen phase, identified by the visible bulb and intact outer root sheath (ORS), were carefully selected under the dissecting microscope. After two rinses, the follicles were transferred into a 35-mm dish. Then the bulge region was amputated from upper follicle by making two transversal cuts respectively at the site of the enlargement spots of ORS with a fine needle. All surgical procedures were operated under a sterile environment. After additional two rinses, the bulge were transferred into a new dish at a density of 40 per dish, immersed in a 3:1 supplemented mixture medium of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12) containing 10% fetal bovine serum as described. The culture were incubated at 37 ◦ C and 5% CO2 in air, and the medium changed twice a week.
Cells plated in 35-mm dish were washed 2× for 5 min with PBS and fixed in ice cold acetone for 10 min. The fixed cells were then washed 3× for 5 min in PBS. Peroxidase and nonspecific antibody binding were blocked by incubation with 1% H2 O2 /methanol and serum for 30 min at room temperature respectively, followed by incubation with antihuman K19 and 1-integrin antibody overnight at 4 ◦ C. The cells were then washed 3× for 5 min to remove unbound primary antibody and incubated with a secondary antibody for 30 min at 37 ◦ C. Unbound second antibody was removed by washing 3× for 5 min, followed by incubating with a third antibody and detected with DAB kit. The cell which showed brown-yellow color in cytoplasm was took as positive cell. Alternatively, the dissected follicles were snap frozen in a −80 ◦ C container and embedded in OCT. Frozen longitudinal sections (8 m) were obtained with a Cryostat (Leica). Whose cutting chamber temperature at −20 ◦ C. The subsequent procedure was carried out as described above. The following antibodies were used: anti-human K19, anti-human 1-integrin, and anti-human K10 (Santa Cruz). Counterstaining with hematoxylin was performed when it was necessary to identify the bulge area.
2.3. Histology
3. Result
In order to observe the morphology of bulge in vivo, the hair follicles were fixed with 4% triformol in PBS. They were then embedded in paraffin and 7 m sections were cut and stained with hematoxylin and eosin.
3.1. Cell culture
2.2. Tissue isolation and cultivation
A modified method described in Section 2 was successfully used to segregate and culture the bulge cells from dissected
52
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 47 (2006) 50–56
Fig. 1. The primary culture of bulge cells from human hair follicles. (A–C) The growth of bulge cells in 2, 3, and 4 days (100×). (D) The bulge cells started to pile up and led to formation of domes (arrowheads) (100×).
human anagen hair follicles in this experiment. The earliest moving-out of cells from the bulge was observed within 2 days after inoculation of bulge block. At the beginning, the cells grew slowly, gathered around the block and became stratified. Then the cells proliferated quickly and formed a round cell sheet (Fig. 1A–C). When reaching a larger size, the bulge cells started to pile up around the block and led to the formation of dome-like cell sheets (Fig. 1D). Subsequently, the outgrowth could be seen along with the edge of the domes, this result indicated that bulge cells retained stronger proliferative capacity in vitro. The cells recovered from liquid nitrogen storage showed no morphologic differences compared with the primary cells, and could proliferate quickly in culture condition. The sizes of the cell sheets incubated in various media were examined (Fig. 2). In DMEM/F12 plus serum mixture, the cell sheets enlarged more quickly and reached the peak at day 5, then kept rapid growth until day 8. In this condition, bulge cells could be successfully cultured and maintained for six passages. The cells recovered from liquid nitrogen storage showed homogeneous epithelium-shape. When WE/serum mixture was used, the cells grew gently and the size of the sheets began to decelerate after day 6. In the serum-free DMEM/F12 mixture, the area of the sheets began to increase at day 4, but the cells proliferated slowly still.
3.2. Morphology of bulge cells Histological examination showed that the bulge was a part of the outer root sheath, which was contiguous with the epidermis. It was located below the opening of the sebaceous duct and serves as the attachment site of arrector pili muscle. The cells in bulge were arranged densely with a uniform, large columnarshape and had a larger nuclear:cytoplasmic ratio (Fig. 3A). These morphological characters were very different from the adjacent epithelial cells.
Fig. 2. The effects of various culture media on cell growth.
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 47 (2006) 50–56
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Fig. 3. (A) The morphologic features of bulge (arrowheads) from human hair follicle (100×). (B and C) The morphologic features of primary bulge cells cultured for 3 days (B) 200×; 2 weeks (C) 400×.
Under light microscopy, the cells in the outgrowth of bulge showed a round and slabstone-shaped appearance with strong refraction. The nuclei of cells were round or oval-shaped and had single and eccentric nucleolus. Mitotic figures were occasionally observed in these cells, showing a proliferative potential in vitro. Longer than 2w in primary culture, the cells far from bulge were morphologically changed into oval-shape. Subsequently, a large number of black granules and vacuoles appeared in the cytoplasm followed by cell crimpling and decreasing (Fig. 3B and C).
integrin and K19, as molecule markers, to identify the cells. Our results showed that the antibodies, K19 and 1-integrin, selectively stained the bulge cells in culture and the positive cells clonally arranged. The proportion of positive cells decreased gradually with long-time culture. The controls were negative entirely (Fig. 5A and B). In the bulge tissues, we also found that 1-integrin and K19 were strongly expressed in situ, while the K10, a marker of differentiated epithelial cell, was not detected in the bulge area (Fig. 6A–C).
3.3. The curve of cell growth The quantity of cultured cells had no change in the first 2 days after inoculation and then the cells proliferated quickly, reaching the peak at the day 6 and maintained in the following days. After 8 days in culture, the cells showed a decelerated tendency in proliferative speed accompanied with a lower cell number (Fig. 4). 3.4. Immunohistochemical staining The identification of stem cells in epidermis and hair follicle by molecular markers remains a controversial discussion. To evaluate the biochemical nature of bulge cells, we chose 1-
Fig. 4. The growth curve of bulge cells.
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Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 47 (2006) 50–56
Fig. 5. Immunohistochemical staining of bulge cells (arrowheads) in primary culture. K19 (A) and 1-integrin (B) (200×).
Fig. 6. Immunohistochemical staining of bulge area (arrowheads) in vivo. K19 (A), 1-integrin (B) and K10 (C) (200×).
4. Discussion Recent data indicated that adult stem cells exist in many tissues, including skeletal muscle, liver, nervous system, epidermis and hematopoietic system. The bulge, as a candidate of habitant of hair stem cell, has been known for many years; however, the limitation of theory and method is still a hindrance in the research progress of this field. Cotsarelis et al. [2], labeling continuously with 3 H-TdR, found that label-retained cells were not observed in the bulb of the pelage, eyelash or vibrissae follicle of mice, but only in the bulge of the upper outer root sheath
(ORS). If pulsing labeling with 3 H-TdR, they found the marks were filled in the matrix, but none in the region of the bulge. So they suggested that follicle stem cell resided in the upper follicle area defined as bulge. More recently, using kinetic and microchimeric techniques, Taylor et al. [4] and Oshima et al. [13] confirmed that the hair follicle bulge was the major repository of skin stem cell. The hair follicle is maintained throughout adult life by stem cell that not only self-renew but generate daughter cells that undergo terminal differentiation. The bulge cells are normally slow-cycling, but can be stimulated to proliferate in response to injuries and to the certain growth stimuli. Moreover,
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 47 (2006) 50–56
the bulge, with pluripotent differentiation potential, is able to not only migrate downwards to produce the matrix keratinocytes of bulb, but also migrate upwards to maintain the epidermis and participate in the formation of hair follicles [5,13,14]. At the present, investigators widely accepted the “Bulge Activation Hypothesis” and the location of HFSC in the bulge, and believed that the HFSC population with a high proliferative potential occurred in the bulge region [2,15,16]. Early in 1980s, Weterings et al. [18] and Wells [17] established primary cultures of human hair follicle keratinocyte respectively. They planted anagen hair follicles and achieved outgrowth of keratinocyte clones. However, the most outgrowths only had rather limited cell numbers and low reproductivity. Especially, many kinds of cellular components were observed in these cultures, and mechanical force put on the plucked hair follicle might make tissues wounded [19,20]. To explore the growth potential and properties of the cells originated from bulge that was a major repository of skin stem cells, after digesting with a modified method, we microdissected hair follicle, precisely cut down and planted the bulge in explant cultures. The advantages of this approach were that the bulge avoided being damaged and the activity of the cells was kept in good condition. Our results showed newly proliferated cells could be observed on the second day after incubation. These cells kept a tremendous proliferative capacity for several days, with a long life span. These phenomena indicated that the cells with high proliferative potential concentrated in the bulge region, which is consistent with several previous reports documenting upper follicle-originated cells displayed higher proliferative capacity and a longer life span than the cells from other regions of hair follicle [21]. Though stem cells play an important role in homeostasis and wound repair, a major obstacle in this field is the lack of special molecular marker. In the skin, Watt [22] found that 1-integrin enriched cells, from both in vitro culture and in vivo tissue, had a higher colony-forming efficiency than native cells, which suggested that 1-integrin was a reliable marker for stem cells. In another studies, Michel et al. [23] described K19 was a better marker than 1-integrin for investigating epithelial stem cell. In previous experiments, the K19 was observed in two places of the skin, the basal layer of epidermis and bulge area of upper hair follicle, where the [3 H] thymidine-label-retaining cells were identified. So the K19 positive cells in skin means epithelial progenitors. In our report, we took 1-integrin and K19 as potential biochemical markers for cutaneous stem cells. Immunohistological staining data showed that 1-integrin and K19 were strongly expressed in bulge area in vivo. The cells in primary culture of bulge also indicated strong expression of 1-integrin and K19, with a descensive tendency in long-time growth, but the differentiation-related keratin K10 was not detected. The reason may be that the bulge cells are apt to differentiate in vitro culture and lose their primitive characteristic. Based on these results of kinetic, morphologic and expressive investigation, our results suggested that bulge-derived cell in vitro culture represented hair follicle stem cells residing in the bulge area in vivo. Because the ORS is contiguous with the bulge region, some investigators presumed that the ORS cells were also stem cells in the hair follicle. But experimental data showed that ORS cells
55
were distinct from bulge cells for the ORS cells: (1) behave a lower colony-forming than bulge cells; (2) represent limited proliferative potential; (3) cannot generate the lower follicle and hair. Our results showed that K19 was negative expressed in the ORS, except bulge area, which was consistent with early study. In recent years, researchers introduced the concept of secondary germ that located in the lower third of hair follicle and contained epithelial stem cells [24]. Ito et al. indicated that the secondary germ was derived form the lowermost portion of the bulge by tracking the labeling cells at anagen onset and following their fate, rather than directly from the ORS [25]. Hair follicles are composed of three kinetically different cell populations. Bulge cells are known as the pluripotent stem cells. The divisions of these slow-cycling stem cells give rise to transit amplifying (TA) cells with decreasing proliferative potential and terminally differentiated (TD) cells with restricted functions. Therefore, the above results imply that the ORS cells are a TA population derived from cell division of bulge cells. As the bulge cells are easily available, these cells can be made an in vitro model for the study of epidermal proliferation and differentiation. Cultured bulge cells are useful not only for in vitro investigations on skin carcinogenesis, but for epidermic and follicular reconstruction, especially in burn healing or serious skin ulcer patients. Meanwhile, the cells might be used to study the influence of drugs on the regulation of hair growth. Further study on the properties of bulge cells will contribute to a better understanding of the mechanism of follicle morphogenesis, cycle maintenance and hair regeneration. Acknowledgement This study was supported by the grants of National Nature Science Foundation of China (Nos. 30371383 and 30471568). References [1] M.C. Lenoir, B.A. Bernard, G. Pautrat, et al., Dev. Biol. 13 (2) (1988) 610–620. [2] G. Cotsarelis, T.T. Sun, R.M. Lavker, Cell 61 (7) (1990) 1329–1337. [3] R.J. Morris, C.S. Potten, J. Invest. Dermatol. 112 (4) (1999) 470–475. [4] G. Taylor, M.S. Lehrer, P.J. Jensen, et al., Cell 102 (4) (2000) 451– 461. [5] R.M. Lavker, S. Miller, C. Wilson, et al., J. Invest. Dermatol. 101 (Suppl. 1) (1993) 16s–26s. [6] R.J. Morris, C.S. Potten, Cell. Prolif. 27 (5) (1994) 279–289. [7] M. Inaba, J. Anthony, C. McKinstry, J. Invest. Dermatol. 72 (5) (1979) 224–231. [8] H. Ura, F. Takeda, H. Okochi, J. Dermatol. Sci. 35 (1) (2004) 19–28. [9] X.H. Lian, T. Yang, Biol. Cell. 96 (2) (2004) 109–116. [10] K. Kobayashi, A. Rochat, Y. Barrandon, Proc. Natl. Acad. Sci. U.S.A. 90 (15) (1993) 7391–7395. [11] A. Rochat, K. Kobayashi, Y. Barrandon, Cell 76 (6) (1994) 1063–1073. [12] J. Kamimura, D. Lee, H.P. Baden, et al., J. Invest. Dermatol. 109 (4) (1997) 534–540. [13] H. Oshima, A. Rochat, C. Kedzia, et al., Cell 104 (2) (2001) 233– 245. [14] A.A. Panteleyev, T. Rosenbach, R. Paus, et al., Arch. Dermatol. Res. 292 (11) (2000) 573–576. [15] E. Fuchs, J.A. Segre, Cell 100 (1) (2000) 143–155. [16] C.S. Potten, C. Booth, J. Invest. Dermatol. 119 (4) (2002) 888–899. [17] J. Wells, Br. J. Dermatol. 107 (4) (1982) 481–482.
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[18] P.J.J.M. Weterings, A.J.M. Vermorken, H. Bloemendal, Br. J. Dermatol. 89 (1987) 369–379. [19] I. Moll, Arch. Dermatol. Res. 288 (10) (1996) 604–610. [20] C.G. Gho, J.E.F. Braun, C.M.L.J. Tilli, et al., Br. J. Dermatol. 150 (5) (2004) 860–868. [21] M. Akiyama, L.T. Smith, H. Shimizu, J. Invest. Dermatol. 114 (2) (2000) 321–327. [22] F.M. Watt, Philos. Trans. Roy. Soc. Lond. B: Biol. Sci. 353 (1370) (1998) 831–837.
[23] M. Michel, N. Torok, M.J. Godbout, et al., J. Cell. Sci. 109 (pt 5) (1996) 1017–1028. [24] S. Commo, O. Gaillard, B.A. Bernard, Differentiation 66 (4–5) (2000) 157–164. [25] M. Ito, K. Kizawa, K. Hamada, et al., Differentiation 72 (9–10) (2004) 548–557. [26] S.E. Al-Barwari, C.S. Potten, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 30 (3) (1976) 201–216.
Bulge cells of human hair follicles: segregation, cultivation and properties Yi Zhang b,1 , Mingming Xiang a , Yun Wang b , Jun Yan b , Yijun Zeng b , Jin Yu b , Tian Yang b,∗ a
Department of Dermatology of Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China b Department of Cell Biology, Third Military Medical University, Chongqing 400038, PR China Received 19 April 2005; received in revised form 12 October 2005; accepted 21 November 2005 Available online 4 January 2006
Abstract The bulge region of hair follicle has been reported as a putative reservoir of hair follicle stem cell (HFSC) for years; however, few studies were done about the characteristics of bulge-originated cells in vitro up to now. In this experiment, the bulge cells isolated from human hair follicles by enzymatic digestion and microdissection were cultured and passaged, and the morphological and biological features of cultured bulge cells were investigated by microscopy and immunocytochemistry. The result showed that new-proliferated cells could be observed on the second day after inoculation, and the quantity of the cells with a greater proliferation potential, reached a peak at the 6th day and maintained this higher level for several days. The mitotic figures of bulge cells were seen and these cells showed undifferentiated morphologic features. The bulge cells strongly expressed K19 and 1-integrin, which are the markers of HFSC, in a descensive way with the culture time. The result indicates that the cultured bulge cell from human hair follicle possesses the properties of primitive cells and supports the hypothesis that HFSC resides in the bulge area. © 2005 Elsevier B.V. All rights reserved. Keywords: Hair follicle; Bulge; Stem cell
1. Introduction The epidermis is the outermost layer of the skin and contacts the external environment directly. It is in continuity with epidermal appendages, such as hair follicle, sebaceous gland and sweat gland, which form through complex epithelio-mesenchymal interactions during embryonic life. Unique to the hair follicle, the epidermis contains a single type of keratinocytes, while hair follicle consists of several different specialized epithelial and connective tissue layers, surrounded by a vascular bed and a neural network. These include three cell types of the hair shaft (medulla, cortex and cuticle), three of inner root sheath (Henle’s and Huxley’s layer, the cuticle) and the outer root sheath. Hair follicle undergoes dynamic changes from an active growing phase (anagen), to a remodeling phase (catagen), and finally to a quiescent phase (telogen). Early in 1954, Chase postulated that the “upper outer root sheath” contains a population of multipotential stem cells capable of not only forming the follicle, but the epidermis and the sebaceous gland. How-
∗ 1
Corresponding author. Tel.: +86 23 68752260; fax: +86 23 65463056. E-mail addresses: [email protected] (Y. Zhang), [email protected] (T. Yang). Tel.: +86 23 68752259.
0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2005.11.017
ever, a major obstacle in this field had been lacking of operative segregation methods and reliable molecular markers. Epidermal appendages have long been known to contribute to hair regeneration and cycle maintenance [1]. It is found that adult stem cells were slow-cycling and self-renewal in vivo, and responsible for the long-term maintenance of the tissues. Many investigators made use of cell kinetics to distinguish the stem cells from the more transit amplifying (TA) cells [2–4]. Al-Barwari and Potten, using [3 H] thymidine to evaluate label retention in murine haired epidermis, discovered that the majority of label-retaining cells (LRCs) in the skin were restricted to the bulge region of the hair follicles [26]. As the bulge represents a slow-cycling population [2,5,6], and may regenerate complete hair follicle after X-irradiation or after removal of the lower parts of the hair follicles [7], it has been proposed that these cells represent multipotent stem cells in the skin, termed as hair follicle stem cell (HFSC). Although more than 10 years has passed since hair follicle bulge was thought to contain stem cells of interfollicular epithelium, the characteristic and the behavior of hair follicle stem cells outside the niche have not been fully understood yet. An extensive knowledge about the bulge is crucial to answer this question. A recent observation in the field of skin biology was that epidermal keratinocytes could be grown in culture and the
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 47 (2006) 50–56
properties of the cells were also discussed [8,9]. Recent years, many investigators were devoted to establish culture of hair follicle cells of mouse, rat and human [10–12]. Growth of human hair keratinocytes in primary culture has been reported using explant culture of plucked hair follicles, but the exact characteristic of cells from bulge region has not been examined in detail. In this report, we described a modified method to harvest intact hair follicles. The bulge, microdissected on the basis of morphologic feature, were cultured and passaged successfully, and then the biologic nature and the expression of markers of bulge cells were also studied. We believe that the work on the characteristic of hair follicle stem cells is essential for understanding follicle regeneration and cycle maintenance. 2. Materials and methods
51
2.4. Subculture and amplification After 2 weeks in primary culture, cells were collected by incubation with a 1:1 mixture of 0.125% trypsin (Sigma) and 0.02% EDTA (Sigma) for 15 min at 37 ◦ C. The dispersed cells were centrifuged for 10 min at 200 × g and replanted in 50 cm2 tissue culture flasks with a medium change every 3–4 days. Cells were routinely passaged every 5–7 days. For the sake of preservation, the cells were digested from culture dishes as described above. After one rinse with culture medium, the cells were resuspended in 1 ml fetal calf serum containing 10% dimethyl sulfoxide (Sigma), transferred into a cryotube, and placed into a freezer in a 4, −20, and −80 ◦ C container by turns. After 24 h the cryotubes were transferred into a liquid nitrogen tank.
2.1. Media 2.5. Determination of growth curve Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (3:1) (Hyclone), supplemented with 10% fetal bovine serum (Hyclone), 10 ng/ml epidermal growth factor (Sigma), hydrocortisone, 5 g/ml insulin (Sigma) and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin). William’E medium (Sigma), also supplemented with 10% fetal bovine serum, 10 ng/ml epidermal growth factor, hydrocortisone, 5 g/ml insulin and antibiotics. Serum-free DMEM/F12 (3:1) was used in some experiments. To examine the effects of various medium conditions on cell growth, we measured the diameter of the cell sheets.
Cells were planted into 4 × 6-well plates at a density of 1 × 104 cells/well. After 2 days in culture, three wells were trypsinized and counted at day 1, 2, 3, 4, 5, 6, 7, 8 and 9 days, respectively. The growth curve was protracted from the mean cell number at each time point. 2.6. Immunohistochemical staining
Plastic surgery specimens of human scalp skin were obtained from the nape of the neck. After rinsing with 70% alcohol, the tissues were trimmed into small pieces (10 mm × 4 mm), and the skin fragments were incubated in 0.25% dispase II for 12 h at 4 ◦ C. The hair follicles were squeezed out carefully. Those in anagen phase, identified by the visible bulb and intact outer root sheath (ORS), were carefully selected under the dissecting microscope. After two rinses, the follicles were transferred into a 35-mm dish. Then the bulge region was amputated from upper follicle by making two transversal cuts respectively at the site of the enlargement spots of ORS with a fine needle. All surgical procedures were operated under a sterile environment. After additional two rinses, the bulge were transferred into a new dish at a density of 40 per dish, immersed in a 3:1 supplemented mixture medium of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12) containing 10% fetal bovine serum as described. The culture were incubated at 37 ◦ C and 5% CO2 in air, and the medium changed twice a week.
Cells plated in 35-mm dish were washed 2× for 5 min with PBS and fixed in ice cold acetone for 10 min. The fixed cells were then washed 3× for 5 min in PBS. Peroxidase and nonspecific antibody binding were blocked by incubation with 1% H2 O2 /methanol and serum for 30 min at room temperature respectively, followed by incubation with antihuman K19 and 1-integrin antibody overnight at 4 ◦ C. The cells were then washed 3× for 5 min to remove unbound primary antibody and incubated with a secondary antibody for 30 min at 37 ◦ C. Unbound second antibody was removed by washing 3× for 5 min, followed by incubating with a third antibody and detected with DAB kit. The cell which showed brown-yellow color in cytoplasm was took as positive cell. Alternatively, the dissected follicles were snap frozen in a −80 ◦ C container and embedded in OCT. Frozen longitudinal sections (8 m) were obtained with a Cryostat (Leica). Whose cutting chamber temperature at −20 ◦ C. The subsequent procedure was carried out as described above. The following antibodies were used: anti-human K19, anti-human 1-integrin, and anti-human K10 (Santa Cruz). Counterstaining with hematoxylin was performed when it was necessary to identify the bulge area.
2.3. Histology
3. Result
In order to observe the morphology of bulge in vivo, the hair follicles were fixed with 4% triformol in PBS. They were then embedded in paraffin and 7 m sections were cut and stained with hematoxylin and eosin.
3.1. Cell culture
2.2. Tissue isolation and cultivation
A modified method described in Section 2 was successfully used to segregate and culture the bulge cells from dissected
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Fig. 1. The primary culture of bulge cells from human hair follicles. (A–C) The growth of bulge cells in 2, 3, and 4 days (100×). (D) The bulge cells started to pile up and led to formation of domes (arrowheads) (100×).
human anagen hair follicles in this experiment. The earliest moving-out of cells from the bulge was observed within 2 days after inoculation of bulge block. At the beginning, the cells grew slowly, gathered around the block and became stratified. Then the cells proliferated quickly and formed a round cell sheet (Fig. 1A–C). When reaching a larger size, the bulge cells started to pile up around the block and led to the formation of dome-like cell sheets (Fig. 1D). Subsequently, the outgrowth could be seen along with the edge of the domes, this result indicated that bulge cells retained stronger proliferative capacity in vitro. The cells recovered from liquid nitrogen storage showed no morphologic differences compared with the primary cells, and could proliferate quickly in culture condition. The sizes of the cell sheets incubated in various media were examined (Fig. 2). In DMEM/F12 plus serum mixture, the cell sheets enlarged more quickly and reached the peak at day 5, then kept rapid growth until day 8. In this condition, bulge cells could be successfully cultured and maintained for six passages. The cells recovered from liquid nitrogen storage showed homogeneous epithelium-shape. When WE/serum mixture was used, the cells grew gently and the size of the sheets began to decelerate after day 6. In the serum-free DMEM/F12 mixture, the area of the sheets began to increase at day 4, but the cells proliferated slowly still.
3.2. Morphology of bulge cells Histological examination showed that the bulge was a part of the outer root sheath, which was contiguous with the epidermis. It was located below the opening of the sebaceous duct and serves as the attachment site of arrector pili muscle. The cells in bulge were arranged densely with a uniform, large columnarshape and had a larger nuclear:cytoplasmic ratio (Fig. 3A). These morphological characters were very different from the adjacent epithelial cells.
Fig. 2. The effects of various culture media on cell growth.
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Fig. 3. (A) The morphologic features of bulge (arrowheads) from human hair follicle (100×). (B and C) The morphologic features of primary bulge cells cultured for 3 days (B) 200×; 2 weeks (C) 400×.
Under light microscopy, the cells in the outgrowth of bulge showed a round and slabstone-shaped appearance with strong refraction. The nuclei of cells were round or oval-shaped and had single and eccentric nucleolus. Mitotic figures were occasionally observed in these cells, showing a proliferative potential in vitro. Longer than 2w in primary culture, the cells far from bulge were morphologically changed into oval-shape. Subsequently, a large number of black granules and vacuoles appeared in the cytoplasm followed by cell crimpling and decreasing (Fig. 3B and C).
integrin and K19, as molecule markers, to identify the cells. Our results showed that the antibodies, K19 and 1-integrin, selectively stained the bulge cells in culture and the positive cells clonally arranged. The proportion of positive cells decreased gradually with long-time culture. The controls were negative entirely (Fig. 5A and B). In the bulge tissues, we also found that 1-integrin and K19 were strongly expressed in situ, while the K10, a marker of differentiated epithelial cell, was not detected in the bulge area (Fig. 6A–C).
3.3. The curve of cell growth The quantity of cultured cells had no change in the first 2 days after inoculation and then the cells proliferated quickly, reaching the peak at the day 6 and maintained in the following days. After 8 days in culture, the cells showed a decelerated tendency in proliferative speed accompanied with a lower cell number (Fig. 4). 3.4. Immunohistochemical staining The identification of stem cells in epidermis and hair follicle by molecular markers remains a controversial discussion. To evaluate the biochemical nature of bulge cells, we chose 1-
Fig. 4. The growth curve of bulge cells.
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Fig. 5. Immunohistochemical staining of bulge cells (arrowheads) in primary culture. K19 (A) and 1-integrin (B) (200×).
Fig. 6. Immunohistochemical staining of bulge area (arrowheads) in vivo. K19 (A), 1-integrin (B) and K10 (C) (200×).
4. Discussion Recent data indicated that adult stem cells exist in many tissues, including skeletal muscle, liver, nervous system, epidermis and hematopoietic system. The bulge, as a candidate of habitant of hair stem cell, has been known for many years; however, the limitation of theory and method is still a hindrance in the research progress of this field. Cotsarelis et al. [2], labeling continuously with 3 H-TdR, found that label-retained cells were not observed in the bulb of the pelage, eyelash or vibrissae follicle of mice, but only in the bulge of the upper outer root sheath
(ORS). If pulsing labeling with 3 H-TdR, they found the marks were filled in the matrix, but none in the region of the bulge. So they suggested that follicle stem cell resided in the upper follicle area defined as bulge. More recently, using kinetic and microchimeric techniques, Taylor et al. [4] and Oshima et al. [13] confirmed that the hair follicle bulge was the major repository of skin stem cell. The hair follicle is maintained throughout adult life by stem cell that not only self-renew but generate daughter cells that undergo terminal differentiation. The bulge cells are normally slow-cycling, but can be stimulated to proliferate in response to injuries and to the certain growth stimuli. Moreover,
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the bulge, with pluripotent differentiation potential, is able to not only migrate downwards to produce the matrix keratinocytes of bulb, but also migrate upwards to maintain the epidermis and participate in the formation of hair follicles [5,13,14]. At the present, investigators widely accepted the “Bulge Activation Hypothesis” and the location of HFSC in the bulge, and believed that the HFSC population with a high proliferative potential occurred in the bulge region [2,15,16]. Early in 1980s, Weterings et al. [18] and Wells [17] established primary cultures of human hair follicle keratinocyte respectively. They planted anagen hair follicles and achieved outgrowth of keratinocyte clones. However, the most outgrowths only had rather limited cell numbers and low reproductivity. Especially, many kinds of cellular components were observed in these cultures, and mechanical force put on the plucked hair follicle might make tissues wounded [19,20]. To explore the growth potential and properties of the cells originated from bulge that was a major repository of skin stem cells, after digesting with a modified method, we microdissected hair follicle, precisely cut down and planted the bulge in explant cultures. The advantages of this approach were that the bulge avoided being damaged and the activity of the cells was kept in good condition. Our results showed newly proliferated cells could be observed on the second day after incubation. These cells kept a tremendous proliferative capacity for several days, with a long life span. These phenomena indicated that the cells with high proliferative potential concentrated in the bulge region, which is consistent with several previous reports documenting upper follicle-originated cells displayed higher proliferative capacity and a longer life span than the cells from other regions of hair follicle [21]. Though stem cells play an important role in homeostasis and wound repair, a major obstacle in this field is the lack of special molecular marker. In the skin, Watt [22] found that 1-integrin enriched cells, from both in vitro culture and in vivo tissue, had a higher colony-forming efficiency than native cells, which suggested that 1-integrin was a reliable marker for stem cells. In another studies, Michel et al. [23] described K19 was a better marker than 1-integrin for investigating epithelial stem cell. In previous experiments, the K19 was observed in two places of the skin, the basal layer of epidermis and bulge area of upper hair follicle, where the [3 H] thymidine-label-retaining cells were identified. So the K19 positive cells in skin means epithelial progenitors. In our report, we took 1-integrin and K19 as potential biochemical markers for cutaneous stem cells. Immunohistological staining data showed that 1-integrin and K19 were strongly expressed in bulge area in vivo. The cells in primary culture of bulge also indicated strong expression of 1-integrin and K19, with a descensive tendency in long-time growth, but the differentiation-related keratin K10 was not detected. The reason may be that the bulge cells are apt to differentiate in vitro culture and lose their primitive characteristic. Based on these results of kinetic, morphologic and expressive investigation, our results suggested that bulge-derived cell in vitro culture represented hair follicle stem cells residing in the bulge area in vivo. Because the ORS is contiguous with the bulge region, some investigators presumed that the ORS cells were also stem cells in the hair follicle. But experimental data showed that ORS cells
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were distinct from bulge cells for the ORS cells: (1) behave a lower colony-forming than bulge cells; (2) represent limited proliferative potential; (3) cannot generate the lower follicle and hair. Our results showed that K19 was negative expressed in the ORS, except bulge area, which was consistent with early study. In recent years, researchers introduced the concept of secondary germ that located in the lower third of hair follicle and contained epithelial stem cells [24]. Ito et al. indicated that the secondary germ was derived form the lowermost portion of the bulge by tracking the labeling cells at anagen onset and following their fate, rather than directly from the ORS [25]. Hair follicles are composed of three kinetically different cell populations. Bulge cells are known as the pluripotent stem cells. The divisions of these slow-cycling stem cells give rise to transit amplifying (TA) cells with decreasing proliferative potential and terminally differentiated (TD) cells with restricted functions. Therefore, the above results imply that the ORS cells are a TA population derived from cell division of bulge cells. As the bulge cells are easily available, these cells can be made an in vitro model for the study of epidermal proliferation and differentiation. Cultured bulge cells are useful not only for in vitro investigations on skin carcinogenesis, but for epidermic and follicular reconstruction, especially in burn healing or serious skin ulcer patients. Meanwhile, the cells might be used to study the influence of drugs on the regulation of hair growth. Further study on the properties of bulge cells will contribute to a better understanding of the mechanism of follicle morphogenesis, cycle maintenance and hair regeneration. Acknowledgement This study was supported by the grants of National Nature Science Foundation of China (Nos. 30371383 and 30471568). References [1] M.C. Lenoir, B.A. Bernard, G. Pautrat, et al., Dev. Biol. 13 (2) (1988) 610–620. [2] G. Cotsarelis, T.T. Sun, R.M. Lavker, Cell 61 (7) (1990) 1329–1337. [3] R.J. Morris, C.S. Potten, J. Invest. Dermatol. 112 (4) (1999) 470–475. [4] G. Taylor, M.S. Lehrer, P.J. Jensen, et al., Cell 102 (4) (2000) 451– 461. [5] R.M. Lavker, S. Miller, C. Wilson, et al., J. Invest. Dermatol. 101 (Suppl. 1) (1993) 16s–26s. [6] R.J. Morris, C.S. Potten, Cell. Prolif. 27 (5) (1994) 279–289. [7] M. Inaba, J. Anthony, C. McKinstry, J. Invest. Dermatol. 72 (5) (1979) 224–231. [8] H. Ura, F. Takeda, H. Okochi, J. Dermatol. Sci. 35 (1) (2004) 19–28. [9] X.H. Lian, T. Yang, Biol. Cell. 96 (2) (2004) 109–116. [10] K. Kobayashi, A. Rochat, Y. Barrandon, Proc. Natl. Acad. Sci. U.S.A. 90 (15) (1993) 7391–7395. [11] A. Rochat, K. Kobayashi, Y. Barrandon, Cell 76 (6) (1994) 1063–1073. [12] J. Kamimura, D. Lee, H.P. Baden, et al., J. Invest. Dermatol. 109 (4) (1997) 534–540. [13] H. Oshima, A. Rochat, C. Kedzia, et al., Cell 104 (2) (2001) 233– 245. [14] A.A. Panteleyev, T. Rosenbach, R. Paus, et al., Arch. Dermatol. Res. 292 (11) (2000) 573–576. [15] E. Fuchs, J.A. Segre, Cell 100 (1) (2000) 143–155. [16] C.S. Potten, C. Booth, J. Invest. Dermatol. 119 (4) (2002) 888–899. [17] J. Wells, Br. J. Dermatol. 107 (4) (1982) 481–482.
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