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Hair Follicle Stem Cell Isolation and Expansion
毛囊干细胞的分离和扩增   

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Stem Cells
Jan 2011

Abstract

Stem cells are widely used for numerous clinical applications including limbal stem cell deficiency. Stem cell derived from the bulge region of the hair follicle have the ability to differentiate into a variety of cell types including interfollicular epidermis, hair follicle structures, sebaceous glands and corneal epithelial cells when provided the appropriate cues. Hair follicle stem cells are being studied as a valuable source of autologous stem cells to treat disease. The protocol described below details the isolation and expansion of these cells for eventual clinical application. We used a dual-reporter mouse model to visualize both isolation and eventual differentiation of these cells in a limbal stem cell-deficient mouse model.

Keywords: Holoclones (全克隆), Clonal expansion (克隆扩增), Hair follicle stem cells (毛囊干细胞), Bulge (毛囊峡部), Stem cell isolation (干细胞分离)

Background

Stem cells are widely used for a multitude of translational and clinical applications. One such clinical application is for the treatment of limbal stem cell deficiency (LSCD). LSCD occurs when there is dysfunction or loss of the limbal stem cell population, which is critical for maintaining a healthy ocular surface, due to congenital or acquired pathologies. The primary treatment strategy for LSCD is cultivating autologous epithelial cell sheets from a limbal biopsy of the patient’s healthy eye (Pellegrini et al., 1997; Shortt et al., 2007). The limitation of this strategy is that it is only applicable for patients that have unilateral LSCD. Those that have bilateral LSCD, must rely on an allogenic limbal biopsy from an immunologically related living donor or cadaveric tissue. Due to the need of systemic immunosuppressive therapy and the limited availability of donor tissue, the therapeutic success rate is decreased. Several research groups have been examining the use of cultivated oral mucosal cells for the treatment of LSCD and have achieved some success. However, these cells often fail to express the corneal epithelial differentiation marker, Keratin 12 (Inatomi et al., 2006) and often result in the development of peripheral neovascularization (Nakamura et al., 2004; Nishida et al., 2004; Ma et al., 2009). Due to these limitations, there was a need for an alternative source of autologous stem cells. Thus we focused on the use of hair follicle stem cells as they harbor multiple sources of stem cells that have been used in regenerative medicine (Cotsarelis et al., 1990; Purba et al., 2014). The hair follicle contains mesenchymal stem cells in the dermal papilla and connective tissue sheath, which can give rise to several cell lineages (Lako et al., 2002; Jahoda et al., 2003; Richardson et al., 2005). Additionally, the bulge region of the hair follicle contains stem cells, which can generate the interfollicular epidermis, hair follicle structures and sebaceous glands (Cotsarelis et al., 1990; Taylor et al., 2000; Cotsarelis, 2006). The hair follicle stem cells (HFSC) derived from the bulge region express of variety of cytokeratins including cytokeratin 15 (Krt15) (Tiede et al., 2007; Kloepper et al., 2008; Larouche et al., 2008), which has been successfully used for the purification and enrichment of HFSC (Blazejewska et al., 2009). HFSC have been successfully used in the treatment of a mouse model of LSCD (Meyer-Blazejewska et al., 2011) and research continues to focus on other therapeutic applications and the eventual translation to humans (Purba et al., 2014). Continued research efforts into these areas rely on a standard method for isolating and expanding the bugle-derived HFSC.

Materials and Reagents

  1. Pipette tips (MidSci, Avant low binding tips)
  2. 35-mm cell culture dish (Thermo Fisher Scientific, catalog number: 153066 )
  3. 6-well plates (Corning, Falcon®, catalog number: 353934 )
  4. 100 mm cell culture dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150464 )
  5. NIH-3T3 cells (ATCC, catalog number: CRL-1658 )
  6. 3-5 week old K12rtTA/rtTA/TetO-Cre/RosamTmG (see Notes)
  7. Ketamine/HCl 100 mg/ml (KetaJect; Henry Schein Animal Health, catalog number: 010177 )
  8. Xylazine AnaSed® 100 mg/ml (Santa Cruz Biotechnology, catalog number: sc-362949Rx )
  9. Collagenase A (Sigma-Aldrich, Roche Diagnostics, catalog number: 10103578001 )
  10. Dispase II (Sigma-Aldrich, catalog number: 4942078001)
    Manufacturer: Roche Diagnostics, catalog number: 04942078001 .
  11. Mitomycin C (Sigma-Aldrich, catalog number: M7949-2MG )
  12. Phosphate buffered saline (PBS)
  13. Trypsin (2.5%) (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
  14. Versene (Thermo Fisher Scientific, GibcoTM, catalog number: 15040066 )
  15. Dulbecco’s modified Eagle medium (DMEM) without calcium and magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 21068028 )
  16. Ham’s F12 Nutrient Mix (Thermo Fisher Scientific, GibcoTM, catalog number: 11765047 )
  17. Fetal Bovine Serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  18. Human recombinant epidermal growth factor (Merck, catalog number: GF144 )
  19. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  20. Calcium Chloride solution 1 M (Sigma-Aldrich, catalog number: 21115 )
  21. Human corneal growth supplement (Thermo Fisher Scientific, GibcoTM, catalog number: S0095 )
  22. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
  23. Amphotericin B (Thermo Fisher Scientific, catalog number: 15290026 )
  24. Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
  25. Stem Cell Media (see Recipes)
  26. 3T3 media (see Recipes)

Equipment

  1. Pipettes
  2. Microdissection scissors (Fine Science Tools, catalog number: 15000-00 )
  3. Forceps (Fine Science Tools, catalog number: 11252-23 )
  4. Scissors (Fine Science Tools, catalog number: 14060-09 )
  5. Hemocytometer (Hausser Scientific, catalog number: 3200 )
  6. Dissecting Scope (ZEISS, model: Stemi DV4 )
  7. BSL2 Laminar flow hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series A2 , catalog number: 1387)
  8. CO2 incubator (Thermo Fisher Scientific, Thermo ScienticTM, model: NAPCO Series 8000 WJ )
  9. Centrifuge (Hettich, model: Rotina 35 )
  10. Inverted fluorescent microscope (Zeiss Observer Z1 with an apotome attachment) (ZEISS, model: AxioObserver Z1 )

Software

  1. AxioVison 4.7
  2. ImageJ

Procedure

  1. Removal of vibrissae (Figure 1)
    1. Sacrifice 3-5 weeks old K12rtTA/rtTA/TetO-Cre/RosamTmG mice with ketamine/xylazine injection followed by cervical dislocation.
    2. Remove the lip pad containing vibrissae with scissors and place in Stem Cell Media.
    3. Under a dissecting microscope, remove the subcutaneous fat and connective tissue with forceps and scissors to expose the rows of vibrissae.
    4. Remove individual vibrissae by pulling away from the pad using fine forceps.


      Figure 1. Isolation of the hair follicle bulge region from K12rtTA/rtTA/TetO-Cre/RosamTmG reporter mice. A. Diagram of the triple transgenic mouse model showing the inducible, tissue-specific nature of the system. B. Scheme showing isolation of the hair follicle bulge region. bg–bulge region. Parts Reprinted with permission from Blazejewska et al., 2009.

  2. Isolation of hair follicle-derived stem cells
    1. Expose the epithelial cores of the vibrissae by cutting the collagen capsule, loosening it from the core and pulling it down along the hair shaft.
    2. Section the epithelial cores into three portions (Figure 2). Place the middle portion containing the hair follicle bulge region into a 35-mm dish containing 2 ml of collagenase (2 mg/ml) and incubate at 37 °C with 5% CO2 for 1 h in order to remove any residual mesenchymal remnants of the capsule.
    3. Transfer the partially digested hair follicle bulge region to another 35-mm dish containing 5 ml dispase/trypsin (2.4 U/0.05%) solution and digest for 1.5 h at 37 °C with 5% CO2 in a humidified chamber to obtain a single-cell suspension of epithelial cells.


      Figure 2. Scheme for the clonal growth assay. The bulge region of the hair follicle is separated via mechanical dissection and enzymatically dissociated. The cells are plated on a 3T3 feeder layer and allowed to form holoclones (white circle at 2 weeks). The red channel depicts the membrane-bound tomato red fluorescence as these cells were derived from the reporter mice described in Figure 1.

  3. Preparing feeder layer
    1. Add Mitomycin C (40 μg/ml) to a 70% confluent dish of NIH3T3 cells and incubate in a humidified chamber at 37 °C with 5% CO2 for 2 h.
    2. Replace the Mitomycin C with 3T3 media.
    3. Trypsinize and seed at 2 x 105 cells per well of a 6-well culture plate.

  4. Expansion/clonal growth assay
    1. Enrich stem and progenitor cells by seeding at 1 x 103 cells/cm2 onto a Mitomycin C inactivated NIH 3T3 feeder layer in a 6-well culture dish (see above).
    2. Cultivate for 14-21 days in a humidified chamber at 37 °C with 5% CO2 to obtain holoclones (Figure 2). Change the medium every 2 days. Cultivate until holoclones are obtained. These will be large colonies containing tightly packed cells.

  5. Subcultivation of hair follicle stem cells
    1. Remove the 3T3 feeder layer with Versene for 60 sec at room temperature.
    2. Wash 2 times with phosphate buffered saline (PBS).
    3. Remove the attached holoclones with trypsin (0.25% Trypsin-EDTA) for 15 min at 37 °C with 5% CO2.
    4. Centrifuge at 170 x g for 5 min.
    5. Cells are ready for application of choice (Note 6).

Data analysis

The conditions provided in this protocol have been optimized to obtain holoclones, which were assessed based on the size of the colony and colony forming efficiency. A detailed analysis of the isolation and clonal expansion of the hair follicle stem cells can be found at Blazejewska et al., 2009.  (Stem Cells 2009 27(3):642-652)

Notes

  1. Perform all cell culture work in a class II biological safety cabinet.
  2. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. Genetically modified mouse lines Krt12rtTA (Chikama et al., 2005), TetO-cre (Perl et al., 2002) and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J(ROSAmTmG) (Muzumdar et al., 2007) have been previously described. Compound transgenic mice were generated by breeding individual mouse lines to create K12rtTA/rtTA/TetO-Cre/RosamTmG. This dual reporter mouse model uses the keratin 12 promoter (corneal epithelium specific) to drive the expression of the Tet-on system. In conjunction with doxycycline and cre, the membrane tomato red hair follicle stem cells will turn green if they have differentiated into corneal epithelial cells.
  3. Take care when removing the hair follicle and exposing the epithelial cores as to not damage the bulge region stem cells with the forceps.
  4. All cell counts were performed using a hemocytometer.
  5. The purity of the hair follicle stem cell cultures could be assessed in a parallel experiment by examining the expression of Krt15.
  6. Bio-protocol title “Murine Hair Follicle Derived Stem Cell Transplantation onto the Cornea Using a Fibrin Carrier” (Call et al., 2018) demonstrates the use of the bulge derived hair follicle stem cells to treat a mouse model of limbal stem cell deficiency.

Recipes

  1. Stem cell media
    3 parts DMEM/High glucose without Ca2+ or Mg2+
    1 part Ham’s F12
    10% FBS
    10 ng/ml EGF
    500 mg/L L-glutamine
    0.4 mM calcium chloride
    1x human corneal growth supplement
    10,000 U/ml penicillin
    10,000 μg/ml streptomycin
    25 μg/ml amphotericin B
  2. 3T3 media
    DMEM-high glucose
    10% FBS
    10,000 U/ml penicillin
    10,000 U/ml streptomycin
    25 μg/ml amphotericin B

Acknowledgments

This study was supported in part by grants from the NIH/NEI EY011845, Ohio Lions Eye Research Foundation to W.W.K. This work was adapted from Blazejewska et al., 2009 in Stem Cells. Authors do not have any conflicts of interest.

References

  1. Blazejewska, E. A., Schlotzer-Schrehardt, U., Zenkel, M., Bachmann, B., Chankiewitz, E., Jacobi, C. and Kruse, F. E. (2009). Corneal limbal microenvironment can induce transdifferentiation of hair follicle stem cells into corneal epithelial-like cells. Stem Cells 27(3): 642-652.
  2. Call, M., Meyer, A. E., Kao, W., Kruse, F. and Schlӧtzer-Schrehardt, U. (2018). Murine hair follicle derived stem cell Transplantation onto the cornea using a fibrin carrier. Bio-protocol 8(10) e2849.
  3. Chikama, T., Hayashi, Y., Liu, C. Y., Terai, N., Terai, K., Kao, C. W., Wang, L., Hayashi, M., Nishida, T., Sanford, P., Doestchman, T. and Kao, W. W. (2005). Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice. Invest Ophthalmol Vis Sci 46(6): 1966-1972.
  4. Cotsarelis, G. (2006). Epithelial stem cells: a folliculocentric view. J Invest Dermatol 126(7): 1459-1468.
  5. Cotsarelis, G., Sun, T. T. and Lavker, R. M. (1990). Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61(7): 1329-1337.
  6. Inatomi, T., Nakamura, T., Koizumi, N., Sotozono, C., Yokoi, N. and Kinoshita, S. (2006). Midterm results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial transplantation. Am J Ophthalmol 141(2): 267-275.
  7. Jahoda, C. A., Whitehouse, J., Reynolds, A. J. and Hole, N. (2003). Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages. Exp Dermatol 12(6): 849-859.
  8. Kloepper, J. E., Tiede, S., Brinckmann, J., Reinhardt, D. P., Meyer, W., Faessler, R. and Paus, R. (2008). Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche. Exp Dermatol 17(7): 592-609.
  9. Lako, M., Armstrong, L., Cairns, P. M., Harris, S., Hole, N. and Jahoda, C. A. (2002). Hair follicle dermal cells repopulate the mouse haematopoietic system. J Cell Sci 115(Pt 20): 3967-3974.
  10. Larouche, D., Tong, X., Fradette, J., Coulombe, P. A. and Germain, L. (2008). Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile. FASEB J 22(5): 1404-1415.
  11. Ma, D. H., Kuo, M. T., Tsai, Y. J., Chen, H. C., Chen, X. L., Wang, S. F., Li, L., Hsiao, C. H. and Lin, K. K. (2009). Transplantation of cultivated oral mucosal epithelial cells for severe corneal burn. Eye (Lond) 23(6): 1442-1450.
  12. Meyer-Blazejewska, E. A., Call, M. K., Yamanaka, O., Liu, H., Schlotzer-Schrehardt, U., Kruse, F. E. and Kao, W. W. (2011). From hair to cornea: toward the therapeutic use of hair follicle-derived stem cells in the treatment of limbal stem cell deficiency. Stem Cells 29(1): 57-66.
  13. Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. and Luo, L. (2007). A global double-fluorescent Cre reporter mouse. Genesis 45(9): 593-605.
  14. Nakamura, T., Inatomi, T., Sotozono, C., Amemiya, T., Kanamura, N. and Kinoshita, S. (2004). Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders. Br J Ophthalmol 88(10): 1280-1284.
  15. Nishida, K., Yamato, M., Hayashida, Y., Watanabe, K., Yamamoto, K., Adachi, E., Nagai, S., Kikuchi, A., Maeda, N., Watanabe, H., Okano, T. and Tano, Y. (2004). Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 351(12): 1187-1196.
  16. Pellegrini, G., Traverso, C. E., Franzi, A. T., Zingirian, M., Cancedda, R. and De Luca, M. (1997). Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349(9057): 990-993.
  17. Perl, A. K., Wert, S. E., Nagy, A., Lobe, C. G. and Whitsett, J. A. (2002). Early restriction of peripheral and proximal cell lineages during formation of the lung. Proc Natl Acad Sci U S A 99(16): 10482-10487.
  18. Purba, T. S., Haslam, I. S., Poblet, E., Jimenez, F., Gandarillas, A., Izeta, A. and Paus, R. (2014). Human epithelial hair follicle stem cells and their progeny: current state of knowledge, the widening gap in translational research and future challenges. Bioessays 36(5): 513-525.
  19. Richardson, G. D., Arnott, E. C., Whitehouse, C. J., Lawrence, C. M., Reynolds, A. J., Hole, N. and Jahoda, C. A. (2005). Plasticity of rodent and human hair follicle dermal cells: implications for cell therapy and tissue engineering. J Investig Dermatol Symp Proc 10(3): 180-183.
  20. Shortt, A. J., Secker, G. A., Notara, M. D., Limb, G. A., Khaw, P. T., Tuft, S. J. and Daniels, J. T. (2007). Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol 52(5): 483-502.
  21. Taylor, G., Lehrer, M. S., Jensen, P. J., Sun, T. T. and Lavker, R. M. (2000). Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102(4): 451-461.
  22. Tiede, S., Kloepper, J. E., Bodo, E., Tiwari, S., Kruse, C. and Paus, R. (2007). Hair follicle stem cells: walking the maze. Eur J Cell Biol 86(7): 355-376.

简介

干细胞被广泛用于许多临床应用,包括角膜缘干细胞缺陷。 当提供适当的提示时,源自毛囊凸出区域的干细胞具有分化成多种细胞类型的能力,包括滤泡间表皮,毛囊结构,皮脂腺和角膜上皮细胞。 正在研究毛囊干细胞作为自体干细胞治疗疾病的宝贵来源。 下面描述的方案详细描述了这些细胞的最终临床应用的分离和扩增。 我们使用双报告小鼠模型来观察这些细胞在角膜缘干细胞缺陷小鼠模型中的分离和最终分化。

【背景】干细胞被广泛用于多种翻译和临床应用。一种这样的临床应用是用于治疗角膜缘干细胞缺陷(LSCD)。当角膜缘干细胞群存在功能障碍或丧失时,LSCD发生,这对于由于先天性或获得性病理而维持健康的眼表非常重要。 LSCD的主要治疗策略是从患者健康眼睛的角膜缘活检组织培养自体上皮细胞片(Pellegrini等人,1997; Shortt等人,2007) 。这种策略的局限性在于它只适用于患有单侧LSCD的患者。那些有双侧LSCD的患者必须依靠免疫相关活体供体或尸体组织的同种异体角膜缘活检。由于全身性免疫抑制治疗的需要和供体组织的有限可用性,治疗成功率降低。一些研究小组一直在研究使用培养的口腔粘膜细胞治疗LSCD并取得了一些成功。然而,这些细胞通常不能表达角膜上皮分化标记角蛋白12(Inatomi等,2006),并且经常导致外周血管新生的发展(Nakamura等人, ,2004; Nishida等人,2004; Ma等人,2009)。由于这些限制,需要自体干细胞的替代来源。因此,我们专注于使用毛囊干细胞,因为它们具有多种再生医学中使用的干细胞来源(Cotsarelis等人,1990; Purba等人, ,2014)。毛囊在真皮乳头和结缔组织鞘中含有间充质干细胞,这可以产生几种细胞谱系(Lako等人,2002; Jahoda等人 ,2003; Richardson等人,2005)。另外,毛囊的隆起区域含有干细胞,其可以产生滤泡间表皮,毛囊结构和皮脂腺(Cotsarelis等人,1990; Taylor等人, 2000; Cotsarelis,2006)。来自凸起区域的毛囊干细胞(HFSC)表达多种细胞角蛋白,包括细胞角蛋白15(Krt15)(Tiede等人,2007; Kloepper等人 ,2008; Larouche等人,2008),其已成功用于纯化和富集HFSC(Blazejewska等人,2009)。 HFSC已成功用于LSCD小鼠模型的治疗(Meyer-Blazejewska et al。,2011),研究继续关注其他治疗应用以及最终向人类的转化(Purba 等人,,2014)。对这些领域的继续研究工作依靠一种标准方法来隔离和扩展军号衍生的HFSC。

关键字:全克隆, 克隆扩增, 毛囊干细胞, 毛囊峡部, 干细胞分离

材料和试剂

  1. 移液器吸头(MidSci,Avant低结合吸头)
  2. 35-mm细胞培养皿(Thermo Fisher Scientific,目录号:153066)
  3. 6孔板(Corning,Falcon ,目录号:353934)
  4. 100mm细胞培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:150464)
  5. NIH-3T3细胞(ATCC,目录号:CRL-1658)
  6. 3-5周龄K12 rtTA / rtTA / TetO-Cre / Rosa mTmG(见注)
  7. 氯胺酮/盐酸100毫克/毫升(KetaJect;亨利沙因动物保健,目录号:010177)
  8. Xylazine AnaSed 100mg / ml(Santa Cruz Biotechnology,目录号:sc-362949Rx)
  9. 胶原酶A(Sigma-Aldrich,Roche Diagnostics,目录号:10103578001)
  10. Dispase II(Sigma-Aldrich,目录号:4942078001)
    制造商:Roche Diagnostics,目录号:04942078001。
  11. 丝裂霉素C(Sigma-Aldrich,目录号:M7949-2MG)
  12. 磷酸盐缓冲盐水(PBS)
  13. 胰蛋白酶(2.5%)(Thermo Fisher Scientific,Gibco TM,目录号:15090046)
  14. Versene(Thermo Fisher Scientific,Gibco TM,目录号:15040066)
  15. Dulbecco改良Eagle培养基(DMEM)不含钙和镁(Thermo Fisher Scientific,Gibco TM,产品目录号:21068028)
  16. Ham's F12营养物混合物(Thermo Fisher Scientific,Gibco TM,目录号:11765047)
  17. 胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:10082147)
  18. 人重组表皮生长因子(Merck,目录号:GF144)
  19. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  20. 氯化钙溶液1M(Sigma-Aldrich,目录号:21115)
  21. 人角膜生长补充剂(Thermo Fisher Scientific,Gibco TM,目录号:S0095)
  22. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140148)
  23. 两性霉素B(Thermo Fisher Scientific,目录号:15290026)
  24. Dulbecco改良的Eagle培养基(DMEM)高葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:11960044)
  25. 干细胞培养基(见食谱)
  26. 3T3媒体(见食谱)

设备

  1. 移液器
  2. 显微切割剪刀(Fine Science Tools,目录编号:15000-00)
  3. 镊子(精细科学工具,目录号:11252-23)
  4. 剪刀(精细科学工具,目录号:14060-09)
  5. 血细胞计数器(Hausser Scientific,目录号:3200)
  6. 解剖镜(蔡司,型号:Stemi DV4)
  7. BSL2层流罩(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300系列A2,目录号:1387)
  8. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientic TM,型号:NAPCO 8000WJ系列)。
  9. 离心机(Hettich,型号:Rotina 35)
  10. 倒置荧光显微镜(蔡司观察员Z1与apotome附件)(蔡司,型号:AxioObserver Z1)

软件

  1. AxioVison 4.7
  2. ImageJ

程序

  1. 去除触须(图1)
    1. 牺牲3-5周龄K12 rtTA / rtTA / TetO-Cre / Rosa mTmG小鼠注射氯胺酮/甲苯噻嗪,然后颈椎脱臼。

    2. 用剪刀取出装有触须的唇垫并置于干细胞培养基中。
    3. 在解剖显微镜下,用镊子和剪刀去除皮下脂肪和结缔组织,暴露出一排排触须。

    4. 使用细镊子从垫子上拉下去除个别触须。


      图1.从K12 rtTA / rtTA / TetO-Cre / Rosa mTmG报告小鼠中分离毛囊凸起区域A.图该三重转基因小鼠模型显示该系统的可诱导的组织特异性质。 B.显示分离毛囊隆起区域的方案。 bg-bulge区域。部件转载于2009年Blazejewska等人的许可,2009年。

  2. 分离毛囊来源的干细胞
    1. 通过切割胶原蛋白胶囊暴露出触须的上皮核心,将其从核心上松开并沿着毛干拉下来。
    2. 将上皮核心切成三部分(图2)。将包含毛囊膨大区域的中间部分放入含有2ml胶原酶(2mg / ml)的35-mm培养皿中,并且在37℃下用5%CO 2孵育1小时依次去除胶囊的任何残余间充质残余物。
    3. 将部分消化的毛囊膨胀区域转移到另一个含有5ml分散酶/胰蛋白酶(2.4U / 0.05%)溶液的35-mm培养皿中,并且在37℃下用5%CO 2 2消化1.5小时一个潮湿的房间,以获得上皮细胞的单细胞悬液。


      图2.用于克隆生长测定的方案通过机械分离和酶分解将毛囊的凸起区域分开。将细胞铺在3T3饲养层上并使其形成holoclone(2周时为白色圆圈)。红色通道描绘了膜结合的番茄红色荧光,因为这些细胞来自图1中描述的报告小鼠。

  3. 准备饲养层
    1. 将丝裂霉素C(40μg/ ml)加入到70%铺满的NIH3T3细胞培养皿中,并在37℃,5%CO 2下潮湿的室中孵育2小时。
    2. 用3T3培养基替换丝裂霉素C.
    3. 胰蛋白酶消化并以每孔2×10 5个细胞接种于6孔培养板中。

  4. 扩增/克隆生长测定
    1. 通过在6孔培养皿中以1×10 3细胞/ cm 2接种到丝裂霉素C灭活的NIH 3T3饲养层上来富集干细胞和祖细胞(见上文) 。
    2. 在37℃和5%CO 2下在潮湿的室中培养14-21天以获得holoclone(图2)。每2天更换一次介质。培养直至获得holoclone。这些将是包含紧密排列的细胞的大型菌落。

  5. 毛囊干细胞的传代培养

    1. 在室温下用Versene去除3T3饲养层60秒。

    2. 用磷酸盐缓冲盐水(PBS)清洗2次
    3. 使用胰蛋白酶(0.25%胰蛋白酶-EDTA)在37°C和5%CO 2下除去连接的holoclones 15分钟。

    4. 170 g x g离心5分钟
    5. 细胞已准备好应用选择(注6)。

数据分析

本协议中提供的条件已经优化以获得holoclone,其基于菌落大小和菌落形成效率进行评估。对毛囊干细胞的分离和克隆扩增的详细分析可以在Blazejewska等人,2009.,Stem Cells 27(3):642-652中找到。 (干细胞2009 27(3):642-652

笔记


  1. 在二级生物安全柜中进行所有细胞培养工作
  2. 所有动物方案均由辛辛那提大学的动物管理和使用委员会批准。转基因小鼠系 Krt12rtTA (Chikama 等人,2005年), TetO-cre (Perl 等人 ,2002)和Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J(ROSAmTmG)(Muzumdar等,<2007>)。通过培养单独的小鼠系来产生化合物转基因小鼠以产生K12 rtTA / rtTA / TetO-Cre / Rosa mTmG。这种双报告小鼠模型使用角蛋白12启动子(角膜上皮特异性)来驱动Tet-on系统的表达。与多西环素和cre联合使用时,膜番茄红毛囊干细胞如果分化为角膜上皮细胞就会变绿。
  3. 去除毛囊和暴露上皮核心时不要用镊子损坏凸起区域干细胞时要小心。
  4. 所有细胞计数均使用血细胞计数器进行。
  5. 通过检查Krt15的表达,可以在平行实验中评估毛囊干细胞培养物的纯度。
  6. 生物协议标题“使用纤维蛋白载体将鼠毛囊衍生的干细胞移植到角膜上”(Call等人,2018)证明了使用凸起衍生的毛囊干细胞来治疗小鼠模型的角膜缘干细胞缺乏。

食谱

  1. 干细胞培养基
    3份DMEM /不含Ca 2 +或Mg 2+ 2+的高葡萄糖 1部分Ham's F12
    10%FBS
    10 ng / ml EGF
    500毫克/升L-谷氨酰胺
    0.4 mM氯化钙
    1x人体角膜生长补充剂
    10,000 U / ml青霉素
    10,000μg/ ml链霉素
    25μg/ ml两性霉素B
  2. 3T3媒体
    DMEM-高葡萄糖
    10%FBS
    10,000 U / ml青霉素
    10,000 U / ml链霉素
    25μg/ ml两性霉素B

致谢

这项研究部分得到了NIH / NEI EY011845,俄亥俄狮子眼科研究基金会和W.W.K的资助。这项工作是从Blazejewska等人改编的,2009年在Stem Cells上发表。作者没有任何利益冲突。

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引用:Call, M., Meyer, E. A., Kao, W. W., Kruse, F. E. and Schloetzer-Schredhardt, U. (2018). Hair Follicle Stem Cell Isolation and Expansion. Bio-protocol 8(10): e2848. DOI: 10.21769/BioProtoc.2848.
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