Characterization and Localization of Side Population Cells in Mouse Skin
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《干细胞学杂志》
a Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, Tokyo;
b Department of Dermatology, Faculty of Medicine, University of Tokyo, Japan
Key Words. Side population ? Stem cell ? Breast cancer resistance protein 1 ? Skin
Correspondence: Hitoshi Okochi, M.D., Ph.D., Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Telephone: 81-3-3202-7181, ext. 2825; Fax: 81-3-3202-7192; e-mail: hokochi@ri.imcj.go.jp
ABSTRACT
Many studies have investigated the localization, character, and function of stem cells in the skin. Multipotent skin stem cells have been thought to be localized in the basal layers or the arrector pili muscle–attaching area in the follicle, called the bulge area, where they have been detected by labeling skin with titrated thymidine or bromodeoxyurindine or colony-forming culture . Several specific molecular markers can distinguish stem cells from other skin cells; 6-integrin, ?1-integrin, CD34, and keratin 19–positive and CD71-negative skin cells are thought to be keratinocyte stem cells . Although many methods have been developed for the isolation and analysis of specific cell types, these cannot be used for medical experimentation on living material because of the damaging techniques, such as isotope radiation and cell fixation.
Recently, it was discovered that hematopoietic stem cells with the characteristics of immature stem cells could be isolated as a specific cell population that had the capability to strongly efflux Hoechst 33342 DNA-binding fluorescent dye . The method relies on incubating the target cells with Hoechst 33342 and performing subsequent fluorescence-activated cell sorter analysis of dual-wavelength Hoechst fluorescence with gating on a specific side population (SP) displaying low red and low blue fluorescence. This low-staining population is called the SP.
These interesting phenomena are explained by the mechanism of a novel stem cell half-transporter. Breast cancer resistance protein (BCRP1), which is one of the multidrug resistance proteins (MDRPs) on the cell membrane and an ATP-binding cassette transporter, predominantly effluxes Hoechst 33342 . MDRPs are associated with resistance to some carcinostatics and are overexpressed in several cancer cell lines . In hematopoietic stem cells, SP cells express relatively high levels of this BCRP1 . Zhou et al. determined that bone marrow SP cells require surface expression of BCRP1 for the efflux of Hoechst dye. Therefore, BCRP1 expression may serve as a new marker for stem cells, not only in hematopoietic cells but also in other types of cells.
As for skin SP cells, only a few studies have been previously performed . It has not been clarified where SP cells are localized and whether skin SP cells possess stem cell characteristics. Accordingly, in the present study, we analyzed skin cells from newborn and adult mice for the presence of SP cells. We further examined the characteristics of the isolated skin SP cells and localized the SPs that were BCRP1-positive in mouse skin.
MATERIALS AND METHODS
SP Cells Were Detected in Mouse Skin
Flow cytometric analysis of skin cell suspensions revealed that approximately 1% of total cells were found distinctly and reproducibly in the tail of the curve (Fig. 2A), representing a side population analogous to bone marrow SP cells (Fig. 2C). This SP was eliminated by verapamil treatment (Figs. 2B, 2D). Interestingly, the frequency of SP cells was relatively higher in mouse skin (approximately 1.4%) than in bone marrow (approximately 0.1%). Cells isolated from the ear skin suspension gave similar results (data not shown). Next, we also measured the SP cells from the epidermis and the dermis separately. SP cells were present at high levels in the epidermis (approximately 5.1%, Fig.2E); however, they were rarely found in the dermis (approximately 0.1%, Fig. 2F), suggesting that SP cells are located mainly in the epidermis in vivo. We then compared the ratios of SP cells among various ages of mice. Epidermal cells from newborn mice had a very high ratio of SP cells. The ratio of SP cells significantly tended to decrease in proportion with aging (Fig. 1).
Figure 2. Detection of side population (SP) cells in mouse skin. Hoechst 33342 staining of a mouse skin cell suspension revealed that (A) approximately 1% of total skin cells showed the SP pattern of staining behavior, which disappeared with (B) verapamil treatment. The gated region suggests SP. The pattern of SP behavior was similar to that of (C, D) bone marrow SP cells. The ratio of SP cells in (E) newborn epidermis was high; however, the ratio of SP cells in (F) newborn dermis was very low.
Characterization of the Sorted SP Cells by Several Markers
To investigate whether the SP cells expressed different surface markers than non-SP cells did, epidermal cells were separately sorted as shown in Figure 3A. We then examined putative stem cell markers. We found that SP cells were more strongly stained with 6-integrin, ?1-integrin, Sca-1, and keratin 14 than non-SP cells (Figs. 3B–3E). Keratin 19 was weakly stained in SP cells but not in non-SP cells (Fig. 3F). On the other hand, we found that SP cells were very weakly stained by CD34, CD71, and E-cadherin compared with non-SP cells (Figs. 3G–3I). Both SP cells and non-SP cells other than epidermal Langerhans cells were negative for CD3, B220 (Figs. 3J, 3K), and CD45RO (Fig. 3L), suggesting that the preparations were not contaminated by hematopoietic cells.
Figure 3. Strong or significant expression of stem cell markers in side population (SP) cells revealed by flow cytometry. SP and non-SP cells were separately sorted (each gate in A). We found that the SP cells (thick line) were more strongly stained by (B) 6-integrin, (C) ?1-integrin, (D) Sca-1, and (E) keratin 14 than non-SP cells (thin line). (F): Keratin 19 weakly stained only SP cells; it did not react with non-SP cells. We also found that SP cells were rarely stained by (G) CD34, (H) CD71, and (I) E-cadherin, but non-SP cells were positively stained. Both SP cells and non-SP cells other than epidermal Langerhans cells were negative for (J) CD3, (K) B220, and (L) CD45RO. Isotype controls are shown as broken lines. Representative results from three sets of independent experiments are shown.
BCRP1 Was Strongly Expressed in SP Cells at Both the Protein and mRNA Levels
Next we examined BCRP1 expression on skin SP cells, because BCRP1 is a key molecule for the SP cell phenotype . After the sorting of both SP and non-SP cells, we analyzed the cell-surface BCRP1 protein. Flow cytometric analysis revealed that BCRP1 was expressed on the cell membrane of skin SP cells more strongly than on non-SP cells (Fig. 4A). Using an anti- BCRP1 monoclonal antibody as a probe during Western blot analysis of sorted cells and tissue lysates, we detected a single protein band with a molecular weight of approximately 72 kDa, the expected size of BCRP1 (Fig. 4B). We found that the SP cells and the epidermis expressed BCRP1; however, non-SP cells and those in the dermis expressed little if any BCRP1. These results suggest that skin SP cells express BCRP1 as do other types of SP cells and that skin SP cells are located mainly in the epidermis, which is consistent with our flow cytometric analysis of epidermal and dermal cell suspensions.
Figure 4. Skin side population (SP) cells strongly express BCRP1 protein and mRNA. (A): Flow cytometric analysis revealed that BCRP1 was more strongly expressed on the cell membranes of skin SP cells (thick line) than on non-SP cells (thin line). (B): Western blot analysis revealed that SP cells and the epidermis expressed BCRP1 and positive control intestine also expressed BCRP1 strongly; however, non-SP cells and dermis did not. Reverse transcription–polymerase chain reaction analysis of SP cells and non-SP cells for BCRP1 and ?-actin (control) was also performed. (C): In adult mice skin, SP cells strongly expressed BCRP1 but non-SP cells showed a weak band. (D): In newborn mice skin, SP cells expressed BCRP1, but non-SP cells, as in the case of adult skin, showed a weak expression. Representative results from three sets of independent experiments are shown.
To further analyze BCRP1 mRNA, RT-PCR was performed. We found that SP cells expressed BCRP1 strongly but non-SP cells in either adult mouse (Fig. 4C) or newborn mouse (Fig. 4D) showed a weak expression. These results suggest that SP cells strongly express both BCRP1 protein on the cell membrane and BCRP1 mRNA.
Localization of BCRP1 in Mouse Skin
Based on the correlation between SP cell and BCRP1 expression, we determined where the BCRP1-positive cells existed. Because BCRP1 is also expressed in blood vessels, we performed CD31 double staining to clarify the location of BCRP1 in the skin. The immunohistochemical staining of newborn mouse skin with anti-mouse BCRP1 and CD31 antibodies combined with the usual staining with hematoxylin and eosin revealed that positive staining was found on the cell membrane in almost all of the basal layers and bulge regions of hair follicles in both newborn (Figs. 5A, 5B) and 1-week-old (Figs. 5C, 5D) mice, consistent with another new report of human skin staining . The skin of adult mouse (Figs. 5E, 5F) stained positively for BCRP1 only in the basal layers and hair bulge regions. The staining intensity of adult skin was therefore relatively weaker than that of newborn skin. The dermal papillae and surrounding lower hair follicle cells did not positively stain in either newborn or adult mice.
Figure 5. Localization of BCRP1 in mouse skin. Immunohistochemical double-staining with anti-BCRP1 antibody and anti-CD31 antibody revealed that BCRP1-positive staining was found in both the basal layers in (A, B) newborn mouse and (C, D) 1-week-old mouse skin. (E, F): Adult mouse skin also showed BCRP1-positive staining in basal layers and hair bulge regions (arrowheads). Endothelium of blood vessels was also stained by BCRP1 antibody, but it was distinguishable as the reactivity to anti-CD31 antibody (red).
We further performed in situ hybridization using BCRP1 mRNA probes. Scattered BCRP1 mRNA expression was detected along the basal layers of the epidermis and hair follicles in newborn mouse skin (Figs. 6A, 6B). No accumulation of positive cells in the bulge regions or dermal papillae was observed. The lung epithelial cells in the newborn mouse were strongly positive, as previously reported (Fig. 6C). In adult mouse skin, BCRP1 mRNA expression was scarcely detected (not shown).
Figure 6. In situ analysis of BCRP1 mRNA. (A, B): In situ hybridization revealed that BCRP1 mRNA expression (arrows) was detected in parts of the basal layers and hair follicles in newborn mouse skin. (C): Lung epithelial cells were used as positive controls (arrows). Scale bar = 100 μm.
Next, we compared the Ki-67 and BCRP1 expression in mouse skin to elucidate the proliferation or self-renewal capacity of the skin SP cells. We found that over half of the BCRP1-positive cells in the basal layer were also positive for Ki-67 (Figs. 7A–7C).
Figure 7. BCRP1-positive cells express Ki-67. Furthermore, over half of the BCRP1-positive cells in the basal layer were positive for Ki-67, which presented in proliferated cells. (A): Anti-Ki-67 antibody-Alexa 594; (B): anti-BCRP1 antibody-Alexa 488; and (C) a merge image of A and B. Scale bar = 100 μm.
DISCUSSION
This work was supported by a Health Science Research Grant from the Japanese Ministry of Health, Labor, and Welfare. The authors thank Dr. Miho Mizukami, Yasuhiko Nagasaka, and Eri Watanabe for technical advice.
REFERENCES
Cotsarelis G, Sun TT, Lavker RM. Label retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle and skin carcinogenesis. Cell 1990;61:1329–1337.
Bickenbach JR, Chism E. Selection and extended growth of murine epidermal stem cells in culture. Exp Cell Res 1998;244:184–195.
Rochat A, Kobayashi K, Barrandon Y. Location of stem cells of human hair follicles by clonal analysis. Cell 1994;76:1063–1073.
Jones PH, Watt FM. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 1993;73:713–724.
Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human epidermis. Cell 1995;80:83–93.
Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A 2000;97:10960–10965.
Michel M, Torok N, Godbout MJ et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 1996;109:1017–1028.
Trempus CS, Morris RJ, Bortner CD et al. Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 2003;120:501–511.
Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.
Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.
Kohno K, Sato S, Takano H et al. The direct activation of human multi-drug resistance gene (MDR1) by anticancer agents. Biochem Biophys Res Commun 1989;165:1415–1421.
Ferguson PJ, Cheng YC. Phenotypic instability of drug sensitivity in a human colon carcinoma cell line. Cancer Res 1989;49:1148–1153.
Kim M, Turnquist H, Jackson J et al. The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 2002;8:22–28.
Zhou S, Morris JJ, Barnes Y et al. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A 2002;99:12339–12344.
Triel C, Vestergaard ME, Bolund L et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res 2004;295:79–90.
Terunuma A, Jackson KL, Kapoor V et al. Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells. J Invest Dermatol 2003;121:1095–1103.
Montanaro F, Liadaki K, Volinski J et al. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc Natl Acad Sci U S A 2003;100:9336–9341.
Montanaro F, Liadaki K, Schienda J et al. Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. Exp Cell Res 2004;298:144–154.
Hoshino M, Sone M, Fukata M et al. Identification of the stef gene that encodes a novel guanine nucleotide exchange factor specific for Rac1. J Biol Chem 1999;274:17837–17844.
Paiva CS, Chen Z, Corrales RM et al. ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. STEM CELLS 2005;23:63–73.
Summer R, Kotton DN, Sun X et al. Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 2003;285:L97–104.
Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci U S A 1999;96:14482–14486.
Alvi AJ, Clayton H, Joshi C et al. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res 2003;5:R1–R8.
Hulspas R, Quesenberry PJ. Characterization of neurosphere cell phenotypes by flow cytometry. Cytometry 2000;40:245–250.
Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507–512.
Pearce DJ, Ridler CM, Simpson C et al. Multiparameter analysis of murine bone marrow side population cells. Blood 2004;103:2541–2546.
Itoh M, Hiraoka Y, Kataoka K et al. Novel collagen sponge reinforced with polyglycolic acid fiber produces robust, normal hair in murine hair reconstitution model. Tissue Eng 2004;10:818–824.
Welm BE, Tepera SB, Venezia T et al. Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 2002;245:42–56.
Purkis PE, Steel JB, Mackenzie IC et al. Antibody markers of basal cells in complex epithelia. J Cell Sci 1990;97:39–50.
Behrens J, Vakaet L, Friis R et al. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J Cell Biol 1993;120:757–766.
Tumbar T, Guasch G, Greco V et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359–363.
Blanpain C, Lowry WE, Geoghegan A et al. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 2004;118:635–648.
Albert MR, Foster RA, Vogel JC. Murine epidermal label-retaining cells isolated by flow cytometry do not express the stem cell markers CD34, Sca-1, or Flk-1. J Invest Dermatol 2001;117:943–948.
Jackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395–1402.(Shoichiro Yanoa,b, Yuriko)
b Department of Dermatology, Faculty of Medicine, University of Tokyo, Japan
Key Words. Side population ? Stem cell ? Breast cancer resistance protein 1 ? Skin
Correspondence: Hitoshi Okochi, M.D., Ph.D., Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. Telephone: 81-3-3202-7181, ext. 2825; Fax: 81-3-3202-7192; e-mail: hokochi@ri.imcj.go.jp
ABSTRACT
Many studies have investigated the localization, character, and function of stem cells in the skin. Multipotent skin stem cells have been thought to be localized in the basal layers or the arrector pili muscle–attaching area in the follicle, called the bulge area, where they have been detected by labeling skin with titrated thymidine or bromodeoxyurindine or colony-forming culture . Several specific molecular markers can distinguish stem cells from other skin cells; 6-integrin, ?1-integrin, CD34, and keratin 19–positive and CD71-negative skin cells are thought to be keratinocyte stem cells . Although many methods have been developed for the isolation and analysis of specific cell types, these cannot be used for medical experimentation on living material because of the damaging techniques, such as isotope radiation and cell fixation.
Recently, it was discovered that hematopoietic stem cells with the characteristics of immature stem cells could be isolated as a specific cell population that had the capability to strongly efflux Hoechst 33342 DNA-binding fluorescent dye . The method relies on incubating the target cells with Hoechst 33342 and performing subsequent fluorescence-activated cell sorter analysis of dual-wavelength Hoechst fluorescence with gating on a specific side population (SP) displaying low red and low blue fluorescence. This low-staining population is called the SP.
These interesting phenomena are explained by the mechanism of a novel stem cell half-transporter. Breast cancer resistance protein (BCRP1), which is one of the multidrug resistance proteins (MDRPs) on the cell membrane and an ATP-binding cassette transporter, predominantly effluxes Hoechst 33342 . MDRPs are associated with resistance to some carcinostatics and are overexpressed in several cancer cell lines . In hematopoietic stem cells, SP cells express relatively high levels of this BCRP1 . Zhou et al. determined that bone marrow SP cells require surface expression of BCRP1 for the efflux of Hoechst dye. Therefore, BCRP1 expression may serve as a new marker for stem cells, not only in hematopoietic cells but also in other types of cells.
As for skin SP cells, only a few studies have been previously performed . It has not been clarified where SP cells are localized and whether skin SP cells possess stem cell characteristics. Accordingly, in the present study, we analyzed skin cells from newborn and adult mice for the presence of SP cells. We further examined the characteristics of the isolated skin SP cells and localized the SPs that were BCRP1-positive in mouse skin.
MATERIALS AND METHODS
SP Cells Were Detected in Mouse Skin
Flow cytometric analysis of skin cell suspensions revealed that approximately 1% of total cells were found distinctly and reproducibly in the tail of the curve (Fig. 2A), representing a side population analogous to bone marrow SP cells (Fig. 2C). This SP was eliminated by verapamil treatment (Figs. 2B, 2D). Interestingly, the frequency of SP cells was relatively higher in mouse skin (approximately 1.4%) than in bone marrow (approximately 0.1%). Cells isolated from the ear skin suspension gave similar results (data not shown). Next, we also measured the SP cells from the epidermis and the dermis separately. SP cells were present at high levels in the epidermis (approximately 5.1%, Fig.2E); however, they were rarely found in the dermis (approximately 0.1%, Fig. 2F), suggesting that SP cells are located mainly in the epidermis in vivo. We then compared the ratios of SP cells among various ages of mice. Epidermal cells from newborn mice had a very high ratio of SP cells. The ratio of SP cells significantly tended to decrease in proportion with aging (Fig. 1).
Figure 2. Detection of side population (SP) cells in mouse skin. Hoechst 33342 staining of a mouse skin cell suspension revealed that (A) approximately 1% of total skin cells showed the SP pattern of staining behavior, which disappeared with (B) verapamil treatment. The gated region suggests SP. The pattern of SP behavior was similar to that of (C, D) bone marrow SP cells. The ratio of SP cells in (E) newborn epidermis was high; however, the ratio of SP cells in (F) newborn dermis was very low.
Characterization of the Sorted SP Cells by Several Markers
To investigate whether the SP cells expressed different surface markers than non-SP cells did, epidermal cells were separately sorted as shown in Figure 3A. We then examined putative stem cell markers. We found that SP cells were more strongly stained with 6-integrin, ?1-integrin, Sca-1, and keratin 14 than non-SP cells (Figs. 3B–3E). Keratin 19 was weakly stained in SP cells but not in non-SP cells (Fig. 3F). On the other hand, we found that SP cells were very weakly stained by CD34, CD71, and E-cadherin compared with non-SP cells (Figs. 3G–3I). Both SP cells and non-SP cells other than epidermal Langerhans cells were negative for CD3, B220 (Figs. 3J, 3K), and CD45RO (Fig. 3L), suggesting that the preparations were not contaminated by hematopoietic cells.
Figure 3. Strong or significant expression of stem cell markers in side population (SP) cells revealed by flow cytometry. SP and non-SP cells were separately sorted (each gate in A). We found that the SP cells (thick line) were more strongly stained by (B) 6-integrin, (C) ?1-integrin, (D) Sca-1, and (E) keratin 14 than non-SP cells (thin line). (F): Keratin 19 weakly stained only SP cells; it did not react with non-SP cells. We also found that SP cells were rarely stained by (G) CD34, (H) CD71, and (I) E-cadherin, but non-SP cells were positively stained. Both SP cells and non-SP cells other than epidermal Langerhans cells were negative for (J) CD3, (K) B220, and (L) CD45RO. Isotype controls are shown as broken lines. Representative results from three sets of independent experiments are shown.
BCRP1 Was Strongly Expressed in SP Cells at Both the Protein and mRNA Levels
Next we examined BCRP1 expression on skin SP cells, because BCRP1 is a key molecule for the SP cell phenotype . After the sorting of both SP and non-SP cells, we analyzed the cell-surface BCRP1 protein. Flow cytometric analysis revealed that BCRP1 was expressed on the cell membrane of skin SP cells more strongly than on non-SP cells (Fig. 4A). Using an anti- BCRP1 monoclonal antibody as a probe during Western blot analysis of sorted cells and tissue lysates, we detected a single protein band with a molecular weight of approximately 72 kDa, the expected size of BCRP1 (Fig. 4B). We found that the SP cells and the epidermis expressed BCRP1; however, non-SP cells and those in the dermis expressed little if any BCRP1. These results suggest that skin SP cells express BCRP1 as do other types of SP cells and that skin SP cells are located mainly in the epidermis, which is consistent with our flow cytometric analysis of epidermal and dermal cell suspensions.
Figure 4. Skin side population (SP) cells strongly express BCRP1 protein and mRNA. (A): Flow cytometric analysis revealed that BCRP1 was more strongly expressed on the cell membranes of skin SP cells (thick line) than on non-SP cells (thin line). (B): Western blot analysis revealed that SP cells and the epidermis expressed BCRP1 and positive control intestine also expressed BCRP1 strongly; however, non-SP cells and dermis did not. Reverse transcription–polymerase chain reaction analysis of SP cells and non-SP cells for BCRP1 and ?-actin (control) was also performed. (C): In adult mice skin, SP cells strongly expressed BCRP1 but non-SP cells showed a weak band. (D): In newborn mice skin, SP cells expressed BCRP1, but non-SP cells, as in the case of adult skin, showed a weak expression. Representative results from three sets of independent experiments are shown.
To further analyze BCRP1 mRNA, RT-PCR was performed. We found that SP cells expressed BCRP1 strongly but non-SP cells in either adult mouse (Fig. 4C) or newborn mouse (Fig. 4D) showed a weak expression. These results suggest that SP cells strongly express both BCRP1 protein on the cell membrane and BCRP1 mRNA.
Localization of BCRP1 in Mouse Skin
Based on the correlation between SP cell and BCRP1 expression, we determined where the BCRP1-positive cells existed. Because BCRP1 is also expressed in blood vessels, we performed CD31 double staining to clarify the location of BCRP1 in the skin. The immunohistochemical staining of newborn mouse skin with anti-mouse BCRP1 and CD31 antibodies combined with the usual staining with hematoxylin and eosin revealed that positive staining was found on the cell membrane in almost all of the basal layers and bulge regions of hair follicles in both newborn (Figs. 5A, 5B) and 1-week-old (Figs. 5C, 5D) mice, consistent with another new report of human skin staining . The skin of adult mouse (Figs. 5E, 5F) stained positively for BCRP1 only in the basal layers and hair bulge regions. The staining intensity of adult skin was therefore relatively weaker than that of newborn skin. The dermal papillae and surrounding lower hair follicle cells did not positively stain in either newborn or adult mice.
Figure 5. Localization of BCRP1 in mouse skin. Immunohistochemical double-staining with anti-BCRP1 antibody and anti-CD31 antibody revealed that BCRP1-positive staining was found in both the basal layers in (A, B) newborn mouse and (C, D) 1-week-old mouse skin. (E, F): Adult mouse skin also showed BCRP1-positive staining in basal layers and hair bulge regions (arrowheads). Endothelium of blood vessels was also stained by BCRP1 antibody, but it was distinguishable as the reactivity to anti-CD31 antibody (red).
We further performed in situ hybridization using BCRP1 mRNA probes. Scattered BCRP1 mRNA expression was detected along the basal layers of the epidermis and hair follicles in newborn mouse skin (Figs. 6A, 6B). No accumulation of positive cells in the bulge regions or dermal papillae was observed. The lung epithelial cells in the newborn mouse were strongly positive, as previously reported (Fig. 6C). In adult mouse skin, BCRP1 mRNA expression was scarcely detected (not shown).
Figure 6. In situ analysis of BCRP1 mRNA. (A, B): In situ hybridization revealed that BCRP1 mRNA expression (arrows) was detected in parts of the basal layers and hair follicles in newborn mouse skin. (C): Lung epithelial cells were used as positive controls (arrows). Scale bar = 100 μm.
Next, we compared the Ki-67 and BCRP1 expression in mouse skin to elucidate the proliferation or self-renewal capacity of the skin SP cells. We found that over half of the BCRP1-positive cells in the basal layer were also positive for Ki-67 (Figs. 7A–7C).
Figure 7. BCRP1-positive cells express Ki-67. Furthermore, over half of the BCRP1-positive cells in the basal layer were positive for Ki-67, which presented in proliferated cells. (A): Anti-Ki-67 antibody-Alexa 594; (B): anti-BCRP1 antibody-Alexa 488; and (C) a merge image of A and B. Scale bar = 100 μm.
DISCUSSION
This work was supported by a Health Science Research Grant from the Japanese Ministry of Health, Labor, and Welfare. The authors thank Dr. Miho Mizukami, Yasuhiko Nagasaka, and Eri Watanabe for technical advice.
REFERENCES
Cotsarelis G, Sun TT, Lavker RM. Label retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle and skin carcinogenesis. Cell 1990;61:1329–1337.
Bickenbach JR, Chism E. Selection and extended growth of murine epidermal stem cells in culture. Exp Cell Res 1998;244:184–195.
Rochat A, Kobayashi K, Barrandon Y. Location of stem cells of human hair follicles by clonal analysis. Cell 1994;76:1063–1073.
Jones PH, Watt FM. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 1993;73:713–724.
Jones PH, Harper S, Watt FM. Stem cell patterning and fate in human epidermis. Cell 1995;80:83–93.
Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A 2000;97:10960–10965.
Michel M, Torok N, Godbout MJ et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 1996;109:1017–1028.
Trempus CS, Morris RJ, Bortner CD et al. Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 2003;120:501–511.
Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.
Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.
Kohno K, Sato S, Takano H et al. The direct activation of human multi-drug resistance gene (MDR1) by anticancer agents. Biochem Biophys Res Commun 1989;165:1415–1421.
Ferguson PJ, Cheng YC. Phenotypic instability of drug sensitivity in a human colon carcinoma cell line. Cancer Res 1989;49:1148–1153.
Kim M, Turnquist H, Jackson J et al. The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 2002;8:22–28.
Zhou S, Morris JJ, Barnes Y et al. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci U S A 2002;99:12339–12344.
Triel C, Vestergaard ME, Bolund L et al. Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res 2004;295:79–90.
Terunuma A, Jackson KL, Kapoor V et al. Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells. J Invest Dermatol 2003;121:1095–1103.
Montanaro F, Liadaki K, Volinski J et al. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc Natl Acad Sci U S A 2003;100:9336–9341.
Montanaro F, Liadaki K, Schienda J et al. Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. Exp Cell Res 2004;298:144–154.
Hoshino M, Sone M, Fukata M et al. Identification of the stef gene that encodes a novel guanine nucleotide exchange factor specific for Rac1. J Biol Chem 1999;274:17837–17844.
Paiva CS, Chen Z, Corrales RM et al. ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. STEM CELLS 2005;23:63–73.
Summer R, Kotton DN, Sun X et al. Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 2003;285:L97–104.
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