Methods in Molecular Biology (2014) 1195: 259–268 DOI 10.1007/7651_2013_61 © Springer Science+Business Media New York 2013 Published online: 5 February 2014
Isolation and Characterization of a Stem Cell Side-Population from Mouse Hair Follicles Paula L. Miliani de Marval, Sun Hye Kim, and Marcelo L. Rodriguez-Puebla Abstract The mouse skin is composed of at least three differentiating epithelial compartments: the epidermis, the hair follicle, and the associated glands such as the sebaceous glands. Proliferation of these epithelial cells takes place in the keratinocytes’ layer or basal cell layer; in the periphery of the sebaceous gland (the basal layer of the gland) and in specific cell compartments around the hair follicle. In mouse skin, an epithelial stem cell population is thought to localize to the bulge region of the hair follicle, a segment that does not undergo regression during the hair cycle. In addition, several other putative stem cells and/or progenitors have been identified in different regions of the hair follicle. Using the Hoeschst exclusion technique, originally described in the hematopoietic system, it has been possible to isolate a mouse keratinocyte cell population with characteristics of stem cells (side-population, SP). One of the main features of these SP is their ability to efflux antimitotic drugs as well as some specific dyes. This characteristic allows for SP cells to be isolated based upon their capacity to efflux the dye Hoechst 33342, through a mechanism driven by a membrane transporter, the breast cancer resistance protein (BCRP1/ABCG2). In this chapter, we described the isolation of SP stem cells from adult mouse hair follicles utilizing the Hoeschst exclusion technique by flow cytometry analysis. Keywords: Side-population, Keratinocyte stem cells, Bulge, Hair follicle, BCRP1/ABCG2, Hoeschst 33342, FACS
1
Introduction Skin stem cells have been identified in the interfollicular epidermis (IFE), the hair follicle bulge region and the sebaceous gland (1–2, 8–10). Numerous molecular markers such as CD34, keratin 15 (K15), Blimp1, and α6 integrin allow distinguishing hair follicle stem cells of the bulge region from keratinocytes (3, 5, 11, 12). In addition, several other putative stem cells and/or progenitors have been identified in different regions of the hair follicle, although their exact role in maintaining epidermal homeostasis and hair follicle morphogenesis is not well understood (4, 13, 14). A subset of cells, termed the side-population (SP), with characteristics of adult stem cells (SCs) has been identified in several
259
260
Paula L. Miliani de Marval et al.
tissues including: mouse epidermis, cell lines, as well as human and experimental tumors (15–17). The main feature of these cell populations is their high efflux capability of antimitotic drugs. This characteristic allows the isolation of the SP based upon their capacity to efflux the dye Hoechst 33342 (6). The membrane transporter “breast cancer resistance protein” (BCRP1/ABCG2), which belongs to the multidrug resistance proteins (MDRPs) family, is responsible for the efflux of Hoechst 33342 (18). In agreement with the high efflux capacity of the SP, high expression of the BCRP1/ABCG2 was observed in the SP from hematopoietic stem cells and other types of cells (19). Remarkably, the SP has characteristics of adult stem cells such as long-term repopulating capacity, undifferentiated phenotype, and colony forming potential (6). However, the role of the SP in tumorigenesis is controversial, though a role as cancer stem cells has been reported (20–22). Therefore, further studies on the role of these putative stem cell populations seems to be necessary to define their main characteristics and differences with adult stem cells and to determine the potential application in regenerative medicine an oncology. In this chapter, we describe a method to isolate SP from the mouse hair follicle. This methodology produces a side-population with characteristics of hair follicle progenitors such as the high expression of the BCRP1/ABCG2 transporter which expression can be confirmed by qRT-PCR.
2 2.1
Materials Solutions
Prepare all the solutions in sterile conditions, under a laminar flow hood. 1. Solution A: 0.25 % Trypsin, without EDTA, without phenol red (Sigma, St. Louis, MO, USA), cool to 4 C. 2. Chelex Serum: Fetal bovine serum (FBS) (Gemini Bioproducts, West Sacramento, CA, USA) chelated with Analytical grade Chelex 100 resin (Cat. # 142-2832, Bio-Rad, Munich, Germany). See Note 1. 3. Solution B: William’s Medium E (GIBCO, Life Technologies, Darmstadt, Germany) + 10 % Chelex serum + Penicillin– Streptomycin (P/S) (Mediatech Inc, Manassas, VA, USA), cool to 4 C. 4. Phosphate-buffered saline (PBS) containing 2 % FBS (chelated), cool to 4 C.
2.2 Instruments and Supplies
1. Sterile dissecting forceps. 2. Sterile dissecting scissors.
Isolation and Characterization of a Stem Cell. . .
261
3. Stainless steel razor blades, Gem Single-Edge Blade (American Safety Razor 94-0451) or #20 Blade stainless steel scalpel. 4. Electric shaver or equivalent. 5. Nair hair remover lotion. 6. Q-tips Cotton-tipped applicators, Single-tipped wood stick applicators (Cat. # NC9586484, Fisher Scientific). 7. 15 and 50-mL polypropylene conical tubes (BD Falcon, Franklin Lakes, NJ). 8. 40-μM and 100-μM nylon cell strainers (Cat. # 08-771-1 and 08-771-19, BD Falcon, Franklin Lakes, NJ). 9. 5-mL cell strainer round-bottom cap tubes (Cat. # 08-771-23, BD Falcon, Franklin Lakes, NJ). 10. Tissue culture dish, 100 20 mm (BD Falcon, Franklin Lakes, NJ). 11. Sterile 50-mL beaker. 12. Sterile stir bar. 13. Laminar flow hood (NuAIR, Plymouth, MN). 14. Sterile and disposable 5-, 10-, and 25-mL tissue culture pipettes (Genesee Scientific, San Diego, CA). 2.3
Reagents
1. Hoechst 33342 (Cat. # B-2261, Sigma, St. Louis, MO). 2. Verapamil (Cat. # V-4629, Sigma, St. Louis, MO). 3. Propidium Iodide (Cat. # P-4170, Sigma, St. Louis, MO).
2.4
3
Mice
1. Three to five 7–9-weeks-old FBV/NCr mice (NCI/Frederick National Laboratory). Isolation of SP can be achieved with mice from any other common laboratory strains. See Note 2.
Methods
3.1 Epidermal Cell Isolation from Mouse Epidermis
Perform all procedures under sterile conditions, under the laminar flow hood preferable. 1. Euthanized mice according to the IACUC approved methods. 2. Shave the entire back of the mouse using an electric shave. Avoid forceful pressure of the shaver against the back of the mouse which could damage the skin. 3. Remove the remaining hair by evenly distributing the Nair depilatory cream on the dorsal skin with a cotton Q-tip or with a Lymtech Clean Room Swabs, (Foam Tip Width 1/200 , Tip Length 100 , Plastic Handle 400 ). Allow the cream to work for 2–3 min.
262
Paula L. Miliani de Marval et al.
Fig. 1 Stepwise procedure for obtaining epidermis from adult mice. (a) Euthanize mice according to laboratory approved procedures, shave dorsal skin, and remove remaining hair with Nair depilatory cream. Dashed dotted line indicates the area of the skin to be removed. (b) Flat skin showing the dermis—shiny side up and fat pads (arrows). (c) Dermis shiny side up after fat removal. (d) Skin cut into 1 1.5 cm to extend the surface exposed to trypsin. (e) Cell culture dish with skins dermis side down floating in trypsin solution (Solution a)
4. Rinse off the Nair lotion and hair thoroughly under running water. 5. Rinse once with 70 % (vol/vol) Ethanol to remove any residual hair (Fig. 1a, b). Pat the skin dry with tissue. 6. Carefully make an incision into the hypodermis with scissors while holding the skin up with forceps and cut off the dorsal skin from the back of the head to right before the beginning of the tail (Fig. 1b, dash line). 7. Dissect the skin in one piece and place it dermal side up (which is the shiny side that contains fat tissue) on a 100-mm tissue culture dish (Fig. 1c). 8. Scrape off all the fat and muscle with a scalpel or razor blade without damaging the underlying epidermis tissue (Fig. 1d).
Isolation and Characterization of a Stem Cell. . .
263
9. Cut the skin into 1 1.5 cm strips using sterile scalpel blade and float the strips with the dermis (shiny side) down on a 100 mm tissue culture dish containing 10-mL of cold Solution A (Fig. 1e, f). Make sure that the epidermis side of the skin does not come into contact with the trypsin. Incubate the floating skin pieces overnight at 4 C or 2 h at 37 C. See Note 3. 10. Prepare 50-mL conical tubes, scalpel blades, forceps, 40-μM and 100-μM nylon cell strainers, 25-mL tissue culture pipettes. After the incubation time with Solution A, perform all the rest of procedures on ice unless otherwise indicated. 11. Place the dish containing the skins floating on Solution A on ice while you process each piece. 12. Transfer one of the floating skin strips from the Solution A plate and place it into a 100 mm microbiology dish, on ice, (do not use tissue culture grade dish since cells tend to adhere to it) with the dermis, shiny side up. Gently scrape off and discard any remaining fat from this side. 13. Flip the skin over to its epidermal side and gently, remove the interfollicular epidermis (opaque side of the tissue) by scraping five times using a scalpel. Discard this material. 14. Move the scraped skin to a new 100 mm microbiology dish containing 2 mL of Solution B, and continue scraping off the epidermis until the shiny dermis is exposed. Discard the scrapped skin strip. 15. Repeat steps #12–14 for the rest of the tissues. Add Solution B as needed. 16. Transfer all the cellular material from all the tissue strips into one small beaker. 17. Pipette the solution up and down at least ten times with a 10-mL tissue culture pipette. 18. Add enough solution B into the beaker containing the cells to complete 25–30 mL total volume. 19. Add a small sterile stir bar and rotate slowly for 30 min at 4 C. 20. Pipette the solution up and down at least ten times with a 10 mL tissue culture pipette. 21. Transfer and filter the solution through a 100-μM nylon cell strainer into a new 50-mL conical tube with a ten tissue culture pipette. 22. Add 5-mL of Solution B to the mesh to wash off any remaining cell. 23. Filter the solution through a 40 μM nylon cell strainer into a new 50-mL conical tube. 24. Spin at 4 C at 200 g in a cell culture centrifuge for 7 min.
264
Paula L. Miliani de Marval et al.
Fig. 2 Phase contrast image of freshly prepared cell fractions from epidermis of adult FBV/NCr mice. Red arrow points to keratinocytes
25. Remove the supernatant carefully and resuspend cells with 1 mL of PBS/2 % Chelex serum, by gently pipetting. 26. Count round-shaped cells in Neubauer chamber (Fig. 2). 27. Adjust the final volume in order to resuspend 1 106 cells per 1 mL of PBS/2 % Chelex serum. 3.2 Epidermal Cell Staining for FACS Analysis
See Tables 1 and 2 for the recommended working samples and dilutions. Hoechst staining 1. Perform a control with verapamil to identify the SP by FACS analysis (Note 4). Incubate a 15-mL conical tube containing 1x106 cells/mL with Verapamil (50 μM final concentration) for 20 min at room temperature, before proceeding with the Hoechst staining. 2. Incubate the verapamil control tube and an additional 15-mL conical tube containing 1 106 cells/mL with 5 μg/mL of Hoechst 33342 at 37 C for 90 min. (Shake tubes frequently during incubation). Note: do not wash the verapamil pretreated tube before incubation with Hoechst 33342. An additional
Isolation and Characterization of a Stem Cell. . .
265
Table 1 Suggested sample set for setup and experiment Sample set
Number of cells
1
Unstained negative control with Propidium Iodide (PI)
1 106
2
Hoechst 33342
1 106
3
Verapamil + Hoechst 33342
1 106
Table 2 Required dye concentrations for cell sample labeling Final concentration (μg/mL) Hoechst 33342 Verapamil Propidium iodide (PI)
5 50 2
control of live cells can be achieved by staining 1 106 cells/mL with add 2 μg/mL of Propidium iodine (PI) (Table 1, sample 1). 3. Add 5-mL cold PBS, and spin stained cells at 200 g, 4 C for 7 min. Discard the supernatant and resuspend the cellular preparation in 200 μL of PBS/2 % FBS Chelex serum (keep cells at 4 C to prohibit leakage of the Hoechst dye). 4. Perform FACS analysis using a DAKO Cytomation MoFlo Ultra-High Speed Cell Sorter or similar system. Set UV laser at 350 nm to excite Hoechst dye and measure using a 450/20nm and 670 band-pass filters (Hoechst blue and red). Cells are analyzed and sorted within PI-negative cells, which represents a living population, and the SP is displayed in a Hoechst blue versus Hoechst red dot plot (Fig. 3a). 5. The side-population gate is chosen by a direct comparison against the verapamil-treated cells. The SP consists of 1–2 % of the total living cells found in the bulge region. The SP and the main population (MP) can be isolated and further analysis of mRNA expression can be performed by qRT-PCR analysis. Figure 3b shows high expression of the ABCG2 transporter in the SP, but no other markers of the bulge stem cells such as CD34, keratin 15 (K15), LRIG1, and integrin α6 are found in this particular population.
266
Paula L. Miliani de Marval et al.
Fig. 3 Identification of side-population (SP) from mouse hair follicles. Dual wavelength FACS analysis shows keratinocytes from the hair follicle incubated with Hoechst 33342. (a) SP cells consist of 1.62 % of total living cells. (b) Total RNA extracted from FACS-sorted side-population (SP) and keratinocyte main-population (MP) were used for qRT-PCR analysis of ABCG2/Bcrp1, CD34, integrin α6, LRIG1, LGR6, Keratin 15 (K15), and CD71. Values >1 represent higher expression on side-population, whereas 1 > values >0 represent low expression in SP compared to keratinocytes MP
4
Notes 1. Removal of calcium from Fetal Bovine Serum using analytical grade 100 Chelex resin, (BioRad Chelex 100 Resin #1422832): Rinse 500 g of resin to remove contaminants through extensive washes by stirring with ddH2O at room temperature by 20–30 min. Stop stirring and allow the resin to settle down for 20 min. Repeat the washes two more times. Resuspend resin in ddH20 and set the pH between 7.0 and 7.4 with HCl. Remove the water from the resin using a Buchner funnel with Whatman paper. Add 2 L of Fetal Bovine Serum to the
Isolation and Characterization of a Stem Cell. . .
267
clean chelex resin and cover with foil. Stir for 4–5 h at 4 C. Stop stirrer and allow chelex to settle. Bring the serum to the cell culture hood and filter through a 500 mL capacity 0.22 μM filter unit. Aliquot serum into 50 mL tubes and store at 20 C. Calcium concentration after chelexing ranges between 20–140 μM. 2. We use 7–9-weeks-old mice during the second resting phase or telogen allowing for more reproducible results. One way to ensure that mice’s hair cycle is indeed in telogen is by clipping their dorsal hair, wait between 48 and 72 h and select those mice with no hair growth (23). 3. Use one 100 mm dish every two mice whole skin. Do not overload the dishes with skins during this step since it will impair the ability of the trypsin to digest the skin properly making it difficult to achieve good isolation of the epidermis from the dermis. Both incubation times (ON at 4 or 2 h at 37 ) work equally well. 4. Verapamil is a prototypical phenylalkylamine that blocks the activity of the ABCG2/BCRP1 transporter allowing gating of the SP by direct comparison with the non-verapamil sample (Table 1).
Acknowledgment This work was supported by National Cancer Institute/NIH grant RO1 CA116328. References 1. Watt FM (1998) Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci 353:831–837 2. Sun TT, Cotsarelis G, Lavker RM (1991) Hair follicular stem cells: the bulge-activation hypothesis. J Invest Dermatol 96:77S–78S 3. Horsley V, O’Carroll D, Tooze R, Ohinata Y, Saitou M, Obukhanych T et al (2006) Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell 126:597–609 4. Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S et al (2009) Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4:427–439 5. Morris RJ, Liu Y, Marles L, Yang Z, Trempus C, Li S et al (2004) Capturing and
profiling adult hair follicle stem cells. Nat Biotechnol 22:411–417 6. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806 7. Dunnwald M, Tomanek-Chalkley A, Alexandrunas D, Fishbaugh J, Bickenbach JR (2001) Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp Dermatol 10:45–54 8. Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61:1329–1337 9. Miller SJ, Burke EM, Rader MD, Coulombe PA, Lavker RM (1998) Re-epithelialization of
268
Paula L. Miliani de Marval et al.
porcine skin by the sweat apparatus. J Invest Dermatol 110:13–19 10. Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM (2000) Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451–461 11. Trempus CS, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, Reece JM et al (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 120:501–511 12. Trempus CS, Morris RJ, Ehinger M, Elmore A, Bortner CD, Ito M et al (2007) CD34 expression by hair follicle stem cells is required for skin tumor development in mice. Cancer Res 67:4173–4181 13. Jensen UB, Yan X, Triel C, Woo SH, Christensen R, Owens DM (2008) A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus. J Cell Sci 121:609–617 14. Nijhof JG, Braun KM, Giangreco A, van Pelt C, Kawamoto H, Boyd RL et al (2006) The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells. Development 133:3027–3037 15. Shimano K, Satake M, Okaya A, Kitanaka J, Kitanaka N, Takemura M et al (2003) Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. Am J Pathol 163:3–9 16. Yano S, Ito Y, Fujimoto M, Hamazaki TS, Tamaki K, Okochi H (2005) Characterization and localization of side population cells in mouse skin. Stem Cells 23:834–841
17. Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24:3–12 18. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ et al (2001) 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 7:1028–1034 19. Kim M, Turnquist H, Jackson J, Sgagias M, Yan Y, Gong M et al (2002) The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 8:22–28 20. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005) Side population is enriched in tumorigenic, stemlike cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res 65:6207–6219 21. Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A et al (2006) Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 44:240–251 22. Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF et al (2006) Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24:506–513 23. Muller-Rover S, Handjiski B, van der Veen C, Eichmuller S, Foitzik K, McKay IA et al (2001) A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 117:3–15
Isolation and Characterization of a Stem Cell Side-Population from Mouse Hair Follicles Paula L. Miliani de Marval, Sun Hye Kim, and Marcelo L. Rodriguez-Puebla Abstract The mouse skin is composed of at least three differentiating epithelial compartments: the epidermis, the hair follicle, and the associated glands such as the sebaceous glands. Proliferation of these epithelial cells takes place in the keratinocytes’ layer or basal cell layer; in the periphery of the sebaceous gland (the basal layer of the gland) and in specific cell compartments around the hair follicle. In mouse skin, an epithelial stem cell population is thought to localize to the bulge region of the hair follicle, a segment that does not undergo regression during the hair cycle. In addition, several other putative stem cells and/or progenitors have been identified in different regions of the hair follicle. Using the Hoeschst exclusion technique, originally described in the hematopoietic system, it has been possible to isolate a mouse keratinocyte cell population with characteristics of stem cells (side-population, SP). One of the main features of these SP is their ability to efflux antimitotic drugs as well as some specific dyes. This characteristic allows for SP cells to be isolated based upon their capacity to efflux the dye Hoechst 33342, through a mechanism driven by a membrane transporter, the breast cancer resistance protein (BCRP1/ABCG2). In this chapter, we described the isolation of SP stem cells from adult mouse hair follicles utilizing the Hoeschst exclusion technique by flow cytometry analysis. Keywords: Side-population, Keratinocyte stem cells, Bulge, Hair follicle, BCRP1/ABCG2, Hoeschst 33342, FACS
1
Introduction Skin stem cells have been identified in the interfollicular epidermis (IFE), the hair follicle bulge region and the sebaceous gland (1–2, 8–10). Numerous molecular markers such as CD34, keratin 15 (K15), Blimp1, and α6 integrin allow distinguishing hair follicle stem cells of the bulge region from keratinocytes (3, 5, 11, 12). In addition, several other putative stem cells and/or progenitors have been identified in different regions of the hair follicle, although their exact role in maintaining epidermal homeostasis and hair follicle morphogenesis is not well understood (4, 13, 14). A subset of cells, termed the side-population (SP), with characteristics of adult stem cells (SCs) has been identified in several
259
260
Paula L. Miliani de Marval et al.
tissues including: mouse epidermis, cell lines, as well as human and experimental tumors (15–17). The main feature of these cell populations is their high efflux capability of antimitotic drugs. This characteristic allows the isolation of the SP based upon their capacity to efflux the dye Hoechst 33342 (6). The membrane transporter “breast cancer resistance protein” (BCRP1/ABCG2), which belongs to the multidrug resistance proteins (MDRPs) family, is responsible for the efflux of Hoechst 33342 (18). In agreement with the high efflux capacity of the SP, high expression of the BCRP1/ABCG2 was observed in the SP from hematopoietic stem cells and other types of cells (19). Remarkably, the SP has characteristics of adult stem cells such as long-term repopulating capacity, undifferentiated phenotype, and colony forming potential (6). However, the role of the SP in tumorigenesis is controversial, though a role as cancer stem cells has been reported (20–22). Therefore, further studies on the role of these putative stem cell populations seems to be necessary to define their main characteristics and differences with adult stem cells and to determine the potential application in regenerative medicine an oncology. In this chapter, we describe a method to isolate SP from the mouse hair follicle. This methodology produces a side-population with characteristics of hair follicle progenitors such as the high expression of the BCRP1/ABCG2 transporter which expression can be confirmed by qRT-PCR.
2 2.1
Materials Solutions
Prepare all the solutions in sterile conditions, under a laminar flow hood. 1. Solution A: 0.25 % Trypsin, without EDTA, without phenol red (Sigma, St. Louis, MO, USA), cool to 4 C. 2. Chelex Serum: Fetal bovine serum (FBS) (Gemini Bioproducts, West Sacramento, CA, USA) chelated with Analytical grade Chelex 100 resin (Cat. # 142-2832, Bio-Rad, Munich, Germany). See Note 1. 3. Solution B: William’s Medium E (GIBCO, Life Technologies, Darmstadt, Germany) + 10 % Chelex serum + Penicillin– Streptomycin (P/S) (Mediatech Inc, Manassas, VA, USA), cool to 4 C. 4. Phosphate-buffered saline (PBS) containing 2 % FBS (chelated), cool to 4 C.
2.2 Instruments and Supplies
1. Sterile dissecting forceps. 2. Sterile dissecting scissors.
Isolation and Characterization of a Stem Cell. . .
261
3. Stainless steel razor blades, Gem Single-Edge Blade (American Safety Razor 94-0451) or #20 Blade stainless steel scalpel. 4. Electric shaver or equivalent. 5. Nair hair remover lotion. 6. Q-tips Cotton-tipped applicators, Single-tipped wood stick applicators (Cat. # NC9586484, Fisher Scientific). 7. 15 and 50-mL polypropylene conical tubes (BD Falcon, Franklin Lakes, NJ). 8. 40-μM and 100-μM nylon cell strainers (Cat. # 08-771-1 and 08-771-19, BD Falcon, Franklin Lakes, NJ). 9. 5-mL cell strainer round-bottom cap tubes (Cat. # 08-771-23, BD Falcon, Franklin Lakes, NJ). 10. Tissue culture dish, 100 20 mm (BD Falcon, Franklin Lakes, NJ). 11. Sterile 50-mL beaker. 12. Sterile stir bar. 13. Laminar flow hood (NuAIR, Plymouth, MN). 14. Sterile and disposable 5-, 10-, and 25-mL tissue culture pipettes (Genesee Scientific, San Diego, CA). 2.3
Reagents
1. Hoechst 33342 (Cat. # B-2261, Sigma, St. Louis, MO). 2. Verapamil (Cat. # V-4629, Sigma, St. Louis, MO). 3. Propidium Iodide (Cat. # P-4170, Sigma, St. Louis, MO).
2.4
3
Mice
1. Three to five 7–9-weeks-old FBV/NCr mice (NCI/Frederick National Laboratory). Isolation of SP can be achieved with mice from any other common laboratory strains. See Note 2.
Methods
3.1 Epidermal Cell Isolation from Mouse Epidermis
Perform all procedures under sterile conditions, under the laminar flow hood preferable. 1. Euthanized mice according to the IACUC approved methods. 2. Shave the entire back of the mouse using an electric shave. Avoid forceful pressure of the shaver against the back of the mouse which could damage the skin. 3. Remove the remaining hair by evenly distributing the Nair depilatory cream on the dorsal skin with a cotton Q-tip or with a Lymtech Clean Room Swabs, (Foam Tip Width 1/200 , Tip Length 100 , Plastic Handle 400 ). Allow the cream to work for 2–3 min.
262
Paula L. Miliani de Marval et al.
Fig. 1 Stepwise procedure for obtaining epidermis from adult mice. (a) Euthanize mice according to laboratory approved procedures, shave dorsal skin, and remove remaining hair with Nair depilatory cream. Dashed dotted line indicates the area of the skin to be removed. (b) Flat skin showing the dermis—shiny side up and fat pads (arrows). (c) Dermis shiny side up after fat removal. (d) Skin cut into 1 1.5 cm to extend the surface exposed to trypsin. (e) Cell culture dish with skins dermis side down floating in trypsin solution (Solution a)
4. Rinse off the Nair lotion and hair thoroughly under running water. 5. Rinse once with 70 % (vol/vol) Ethanol to remove any residual hair (Fig. 1a, b). Pat the skin dry with tissue. 6. Carefully make an incision into the hypodermis with scissors while holding the skin up with forceps and cut off the dorsal skin from the back of the head to right before the beginning of the tail (Fig. 1b, dash line). 7. Dissect the skin in one piece and place it dermal side up (which is the shiny side that contains fat tissue) on a 100-mm tissue culture dish (Fig. 1c). 8. Scrape off all the fat and muscle with a scalpel or razor blade without damaging the underlying epidermis tissue (Fig. 1d).
Isolation and Characterization of a Stem Cell. . .
263
9. Cut the skin into 1 1.5 cm strips using sterile scalpel blade and float the strips with the dermis (shiny side) down on a 100 mm tissue culture dish containing 10-mL of cold Solution A (Fig. 1e, f). Make sure that the epidermis side of the skin does not come into contact with the trypsin. Incubate the floating skin pieces overnight at 4 C or 2 h at 37 C. See Note 3. 10. Prepare 50-mL conical tubes, scalpel blades, forceps, 40-μM and 100-μM nylon cell strainers, 25-mL tissue culture pipettes. After the incubation time with Solution A, perform all the rest of procedures on ice unless otherwise indicated. 11. Place the dish containing the skins floating on Solution A on ice while you process each piece. 12. Transfer one of the floating skin strips from the Solution A plate and place it into a 100 mm microbiology dish, on ice, (do not use tissue culture grade dish since cells tend to adhere to it) with the dermis, shiny side up. Gently scrape off and discard any remaining fat from this side. 13. Flip the skin over to its epidermal side and gently, remove the interfollicular epidermis (opaque side of the tissue) by scraping five times using a scalpel. Discard this material. 14. Move the scraped skin to a new 100 mm microbiology dish containing 2 mL of Solution B, and continue scraping off the epidermis until the shiny dermis is exposed. Discard the scrapped skin strip. 15. Repeat steps #12–14 for the rest of the tissues. Add Solution B as needed. 16. Transfer all the cellular material from all the tissue strips into one small beaker. 17. Pipette the solution up and down at least ten times with a 10-mL tissue culture pipette. 18. Add enough solution B into the beaker containing the cells to complete 25–30 mL total volume. 19. Add a small sterile stir bar and rotate slowly for 30 min at 4 C. 20. Pipette the solution up and down at least ten times with a 10 mL tissue culture pipette. 21. Transfer and filter the solution through a 100-μM nylon cell strainer into a new 50-mL conical tube with a ten tissue culture pipette. 22. Add 5-mL of Solution B to the mesh to wash off any remaining cell. 23. Filter the solution through a 40 μM nylon cell strainer into a new 50-mL conical tube. 24. Spin at 4 C at 200 g in a cell culture centrifuge for 7 min.
264
Paula L. Miliani de Marval et al.
Fig. 2 Phase contrast image of freshly prepared cell fractions from epidermis of adult FBV/NCr mice. Red arrow points to keratinocytes
25. Remove the supernatant carefully and resuspend cells with 1 mL of PBS/2 % Chelex serum, by gently pipetting. 26. Count round-shaped cells in Neubauer chamber (Fig. 2). 27. Adjust the final volume in order to resuspend 1 106 cells per 1 mL of PBS/2 % Chelex serum. 3.2 Epidermal Cell Staining for FACS Analysis
See Tables 1 and 2 for the recommended working samples and dilutions. Hoechst staining 1. Perform a control with verapamil to identify the SP by FACS analysis (Note 4). Incubate a 15-mL conical tube containing 1x106 cells/mL with Verapamil (50 μM final concentration) for 20 min at room temperature, before proceeding with the Hoechst staining. 2. Incubate the verapamil control tube and an additional 15-mL conical tube containing 1 106 cells/mL with 5 μg/mL of Hoechst 33342 at 37 C for 90 min. (Shake tubes frequently during incubation). Note: do not wash the verapamil pretreated tube before incubation with Hoechst 33342. An additional
Isolation and Characterization of a Stem Cell. . .
265
Table 1 Suggested sample set for setup and experiment Sample set
Number of cells
1
Unstained negative control with Propidium Iodide (PI)
1 106
2
Hoechst 33342
1 106
3
Verapamil + Hoechst 33342
1 106
Table 2 Required dye concentrations for cell sample labeling Final concentration (μg/mL) Hoechst 33342 Verapamil Propidium iodide (PI)
5 50 2
control of live cells can be achieved by staining 1 106 cells/mL with add 2 μg/mL of Propidium iodine (PI) (Table 1, sample 1). 3. Add 5-mL cold PBS, and spin stained cells at 200 g, 4 C for 7 min. Discard the supernatant and resuspend the cellular preparation in 200 μL of PBS/2 % FBS Chelex serum (keep cells at 4 C to prohibit leakage of the Hoechst dye). 4. Perform FACS analysis using a DAKO Cytomation MoFlo Ultra-High Speed Cell Sorter or similar system. Set UV laser at 350 nm to excite Hoechst dye and measure using a 450/20nm and 670 band-pass filters (Hoechst blue and red). Cells are analyzed and sorted within PI-negative cells, which represents a living population, and the SP is displayed in a Hoechst blue versus Hoechst red dot plot (Fig. 3a). 5. The side-population gate is chosen by a direct comparison against the verapamil-treated cells. The SP consists of 1–2 % of the total living cells found in the bulge region. The SP and the main population (MP) can be isolated and further analysis of mRNA expression can be performed by qRT-PCR analysis. Figure 3b shows high expression of the ABCG2 transporter in the SP, but no other markers of the bulge stem cells such as CD34, keratin 15 (K15), LRIG1, and integrin α6 are found in this particular population.
266
Paula L. Miliani de Marval et al.
Fig. 3 Identification of side-population (SP) from mouse hair follicles. Dual wavelength FACS analysis shows keratinocytes from the hair follicle incubated with Hoechst 33342. (a) SP cells consist of 1.62 % of total living cells. (b) Total RNA extracted from FACS-sorted side-population (SP) and keratinocyte main-population (MP) were used for qRT-PCR analysis of ABCG2/Bcrp1, CD34, integrin α6, LRIG1, LGR6, Keratin 15 (K15), and CD71. Values >1 represent higher expression on side-population, whereas 1 > values >0 represent low expression in SP compared to keratinocytes MP
4
Notes 1. Removal of calcium from Fetal Bovine Serum using analytical grade 100 Chelex resin, (BioRad Chelex 100 Resin #1422832): Rinse 500 g of resin to remove contaminants through extensive washes by stirring with ddH2O at room temperature by 20–30 min. Stop stirring and allow the resin to settle down for 20 min. Repeat the washes two more times. Resuspend resin in ddH20 and set the pH between 7.0 and 7.4 with HCl. Remove the water from the resin using a Buchner funnel with Whatman paper. Add 2 L of Fetal Bovine Serum to the
Isolation and Characterization of a Stem Cell. . .
267
clean chelex resin and cover with foil. Stir for 4–5 h at 4 C. Stop stirrer and allow chelex to settle. Bring the serum to the cell culture hood and filter through a 500 mL capacity 0.22 μM filter unit. Aliquot serum into 50 mL tubes and store at 20 C. Calcium concentration after chelexing ranges between 20–140 μM. 2. We use 7–9-weeks-old mice during the second resting phase or telogen allowing for more reproducible results. One way to ensure that mice’s hair cycle is indeed in telogen is by clipping their dorsal hair, wait between 48 and 72 h and select those mice with no hair growth (23). 3. Use one 100 mm dish every two mice whole skin. Do not overload the dishes with skins during this step since it will impair the ability of the trypsin to digest the skin properly making it difficult to achieve good isolation of the epidermis from the dermis. Both incubation times (ON at 4 or 2 h at 37 ) work equally well. 4. Verapamil is a prototypical phenylalkylamine that blocks the activity of the ABCG2/BCRP1 transporter allowing gating of the SP by direct comparison with the non-verapamil sample (Table 1).
Acknowledgment This work was supported by National Cancer Institute/NIH grant RO1 CA116328. References 1. Watt FM (1998) Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci 353:831–837 2. Sun TT, Cotsarelis G, Lavker RM (1991) Hair follicular stem cells: the bulge-activation hypothesis. J Invest Dermatol 96:77S–78S 3. Horsley V, O’Carroll D, Tooze R, Ohinata Y, Saitou M, Obukhanych T et al (2006) Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell 126:597–609 4. Jensen KB, Collins CA, Nascimento E, Tan DW, Frye M, Itami S et al (2009) Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4:427–439 5. Morris RJ, Liu Y, Marles L, Yang Z, Trempus C, Li S et al (2004) Capturing and
profiling adult hair follicle stem cells. Nat Biotechnol 22:411–417 6. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806 7. Dunnwald M, Tomanek-Chalkley A, Alexandrunas D, Fishbaugh J, Bickenbach JR (2001) Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp Dermatol 10:45–54 8. Cotsarelis G, Sun TT, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61:1329–1337 9. Miller SJ, Burke EM, Rader MD, Coulombe PA, Lavker RM (1998) Re-epithelialization of
268
Paula L. Miliani de Marval et al.
porcine skin by the sweat apparatus. J Invest Dermatol 110:13–19 10. Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM (2000) Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451–461 11. Trempus CS, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, Reece JM et al (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 120:501–511 12. Trempus CS, Morris RJ, Ehinger M, Elmore A, Bortner CD, Ito M et al (2007) CD34 expression by hair follicle stem cells is required for skin tumor development in mice. Cancer Res 67:4173–4181 13. Jensen UB, Yan X, Triel C, Woo SH, Christensen R, Owens DM (2008) A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus. J Cell Sci 121:609–617 14. Nijhof JG, Braun KM, Giangreco A, van Pelt C, Kawamoto H, Boyd RL et al (2006) The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells. Development 133:3027–3037 15. Shimano K, Satake M, Okaya A, Kitanaka J, Kitanaka N, Takemura M et al (2003) Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. Am J Pathol 163:3–9 16. Yano S, Ito Y, Fujimoto M, Hamazaki TS, Tamaki K, Okochi H (2005) Characterization and localization of side population cells in mouse skin. Stem Cells 23:834–841
17. Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24:3–12 18. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ et al (2001) 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 7:1028–1034 19. Kim M, Turnquist H, Jackson J, Sgagias M, Yan Y, Gong M et al (2002) The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 8:22–28 20. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005) Side population is enriched in tumorigenic, stemlike cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res 65:6207–6219 21. Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A et al (2006) Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 44:240–251 22. Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF et al (2006) Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24:506–513 23. Muller-Rover S, Handjiski B, van der Veen C, Eichmuller S, Foitzik K, McKay IA et al (2001) A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 117:3–15
