人多潜能毛囊干细胞的分离培养及其向神经元样细胞诱导分化的研究
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摘要
疾病对人类的健康和生存构成重大威胁、是世界各国普遍面临的最重要的社会问题。干细胞移植技术的问世和进展为传统治疗手段认为是“不治之症”的患者带来了新的生机和希望。但干细胞的来源、自我更新潜能的维持、向功能细胞分化的潜能以及移植治疗的安全性、有效性一直是干细胞基础理论和实际应用研究中急待解决的关键科学问题。
毛囊是皮肤的附属器之一,起源于表皮与间充质间的相互作用,终生自我更新,是存在于成体内的胚胎样器官。新近研究发现,毛囊中除了含有表皮干细胞和黑色素干细胞外,还含有具有多向分化潜能的间充质干细胞。这些多潜能干细胞具有自我更新和定向分化成其它细胞的能力。与其他类型间充质干细胞相比,毛囊干细胞具有来源丰富,获取方便,不受年龄性别限制,不涉及伦理问题等优点。
首先,本研究探索了一种简便可行的获取毛囊多潜能干细胞的方法,通过应用组织块培养法,从拔取的人毛发中分离培养出多潜能毛囊干细胞,并应用免疫细胞化学法结合流式细胞仪技术,对毛囊多潜能干细胞的表型进行了分析鉴定,结果显示多潜能毛囊干细胞表达间充质干细胞相对特异的表面标记物CD29、CD44、CD90、CD105(SH-2)、CD73(SH-4)表面抗原,不表达CD31、CD34、CD45、HLA-DR、K15等表面抗原。其次,本实验初步建立了毛囊多潜能干细胞传代、扩增、冻存与复苏的方法。最后,本实验成功诱导多潜能毛囊干细胞向成骨细胞、脂肪细胞分化。探索了多潜能毛囊干细胞向神经样细胞诱导分化的条件,通过免疫细胞化学法检测诱导前后神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-L)、胶质纤维酸性蛋白(GFAP)、神经巢蛋白(nestin)、神经微管蛋白Ⅲ(β-tubulinⅢ)、S100钙结合蛋白B(S100B)及环核苷磷酸二酯酶(CNPase)的表达水平。认为双丁酰环磷腺苷(Dibutyryl Cyclic AMP)联合3-异丁基-1-甲基黄嘌呤(IBMX)是目前本实验条件下诱导人毛囊干细胞向神经元细胞分化最为有效的方法。为后期利用多潜能毛囊干细胞移植修复损伤神经、帕金森及其它神经系统病变奠定了必要的前期工作基础。
The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection.
Recent researches found that hair follicle contains a variety of stem cells e×cepts epidermal stem cells and melanoma cells . hair follicle pluripotent stem cells present in the hair follicle dermal papilla and dermal sheath with self-renewal ability and differentiation potential. under certain condition cells can differentiate into a variety of cells. The cells in the adult dermal papilla and dermal sheath are well-characterised particularly because of their remarkable inductive powers.Directly isolated and cultured dermal cells from mature adult follicles have been shown to be capable of inducing a range of epithelia to form follicles.
multipotent human follicular stem cells in the body with slow periodic, but have high proliferative capacity specific in vitro conditions. multipotent human follicular stem cells have self-renewal ability and differentiation potential, under certain conditions cells can differentiate into a variety of functions. multipotent differentiation capacity is the most important characteristic to the identification of stem cells. More and more research have proved that mesenchymal stem cells can different into a variety of cell by given the cultivation of different cell differentiation conditions.
Our e×periment is studying on the multipotent human follicular stem cells’segregation, culture and identification. In e×periment, we obtained the human hair follicle-derived mesenchymal-like stem cells simply by plucking the adults’hair . The flow cytometry analysis shows their high e×pression of mesenchymal stem cells related markers , including: CD29、CD44、CD73、CD90、CD105;rare e×pression of epidermal stem cells marker CK15, hematopoietic stem cells marker CD34,leukocyte marker CD45,endothelial cell marker CD31 and immune response-related major histocompatibility antigen HLA-DR.All the above indicates that these cells are rich in mesenchymal cells. The multipotent human follicular stem cells showed adipogenic and osteogenic capacity and can different into neuronal-like cells with db-cAMP and IBMX. So multipotent human follicular stem cells may treat spinal cord injury, Parkinson's disease and other nervous system disease one day.
毛囊是皮肤的附属器之一,起源于表皮与间充质间的相互作用,终生自我更新,是存在于成体内的胚胎样器官。新近研究发现,毛囊中除了含有表皮干细胞和黑色素干细胞外,还含有具有多向分化潜能的间充质干细胞。这些多潜能干细胞具有自我更新和定向分化成其它细胞的能力。与其他类型间充质干细胞相比,毛囊干细胞具有来源丰富,获取方便,不受年龄性别限制,不涉及伦理问题等优点。
首先,本研究探索了一种简便可行的获取毛囊多潜能干细胞的方法,通过应用组织块培养法,从拔取的人毛发中分离培养出多潜能毛囊干细胞,并应用免疫细胞化学法结合流式细胞仪技术,对毛囊多潜能干细胞的表型进行了分析鉴定,结果显示多潜能毛囊干细胞表达间充质干细胞相对特异的表面标记物CD29、CD44、CD90、CD105(SH-2)、CD73(SH-4)表面抗原,不表达CD31、CD34、CD45、HLA-DR、K15等表面抗原。其次,本实验初步建立了毛囊多潜能干细胞传代、扩增、冻存与复苏的方法。最后,本实验成功诱导多潜能毛囊干细胞向成骨细胞、脂肪细胞分化。探索了多潜能毛囊干细胞向神经样细胞诱导分化的条件,通过免疫细胞化学法检测诱导前后神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-L)、胶质纤维酸性蛋白(GFAP)、神经巢蛋白(nestin)、神经微管蛋白Ⅲ(β-tubulinⅢ)、S100钙结合蛋白B(S100B)及环核苷磷酸二酯酶(CNPase)的表达水平。认为双丁酰环磷腺苷(Dibutyryl Cyclic AMP)联合3-异丁基-1-甲基黄嘌呤(IBMX)是目前本实验条件下诱导人毛囊干细胞向神经元细胞分化最为有效的方法。为后期利用多潜能毛囊干细胞移植修复损伤神经、帕金森及其它神经系统病变奠定了必要的前期工作基础。
The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection.
Recent researches found that hair follicle contains a variety of stem cells e×cepts epidermal stem cells and melanoma cells . hair follicle pluripotent stem cells present in the hair follicle dermal papilla and dermal sheath with self-renewal ability and differentiation potential. under certain condition cells can differentiate into a variety of cells. The cells in the adult dermal papilla and dermal sheath are well-characterised particularly because of their remarkable inductive powers.Directly isolated and cultured dermal cells from mature adult follicles have been shown to be capable of inducing a range of epithelia to form follicles.
multipotent human follicular stem cells in the body with slow periodic, but have high proliferative capacity specific in vitro conditions. multipotent human follicular stem cells have self-renewal ability and differentiation potential, under certain conditions cells can differentiate into a variety of functions. multipotent differentiation capacity is the most important characteristic to the identification of stem cells. More and more research have proved that mesenchymal stem cells can different into a variety of cell by given the cultivation of different cell differentiation conditions.
Our e×periment is studying on the multipotent human follicular stem cells’segregation, culture and identification. In e×periment, we obtained the human hair follicle-derived mesenchymal-like stem cells simply by plucking the adults’hair . The flow cytometry analysis shows their high e×pression of mesenchymal stem cells related markers , including: CD29、CD44、CD73、CD90、CD105;rare e×pression of epidermal stem cells marker CK15, hematopoietic stem cells marker CD34,leukocyte marker CD45,endothelial cell marker CD31 and immune response-related major histocompatibility antigen HLA-DR.All the above indicates that these cells are rich in mesenchymal cells. The multipotent human follicular stem cells showed adipogenic and osteogenic capacity and can different into neuronal-like cells with db-cAMP and IBMX. So multipotent human follicular stem cells may treat spinal cord injury, Parkinson's disease and other nervous system disease one day.
引文
[1] Amoh Y, Kanoh M, Niiyama S, et al. Human Hair Follicle Pluripotent Stem Cells Promote Regeneration of Peripheral-Nerve Injury: An Advantageous Alternative to ES and iPS Cells.[J]Journal of Cellular Biochemistry,2009, 107:1016–1020
[2] Seeberger KL, Dufour JM, Shapiro AM, et al. Expansion of mesenchymal stem cells from human pancreatic ductal epithelium. [ J ] Lab Invest 2006; 86(2):141-153.
[3] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. [J]Natrue 2002; 418(6893):41-49.
[4] Amoh Y, Li L, Katsuoka K, et al. Multipotentnestin positive, keratin-negative hair-follicle bulge stem cells can form neurons. [J]PNAS, 2005, 102(15):5530-5534.
[5] Lavker R, Sun T, Oshima H. et al. Hair follicle stem cells. J. Invest. Dermatol. Symp. Proc. 8, 28-38. (2003).
[6] Millar, S. E. Molecular mechanisms regulating hair follicle development. J. Invest. Dermatol. 2002,118, 216-225.
[7] Muller R, Handjiski B, vanderVeen. et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J. Invest. Dermatol. 117, 3-15.
[8] R Paus, G Cotsarelis .The Biology of Hair Follicles. [J]The New England Journal of Medicine.1999,Volume 341:491-497
[9] SchmidtUllrich, R. Paus, R. Molecular principles of hair follicle induction and morphogenesis. [J]BioEssays.2005 ,27, 247-261.
[10] Jahoda CA., Horne KA Oliver RF. Induction of hair growth by implantation of cultured dermal papilla cells. [J]Nature.1984 ,311, 560-562.
[11] Kishimoto J, Burgeson RE and Morgan BA. Wnt signaling maintains the hair-inducing activity of the dermal papilla. [J]Genes Dev. 2000 ,14, 1181-1185.
[12] Alonso L, Fuchs E.The hair cycle. Journal of Cell Science .2006, 119, 391-393
[13] Chuong CM. Molecular basis of epithelial appendage morphogenesis. [D]Molecular biology intelligence unit 1. Austin, Tex.: R.G. Landes, 1998.
[14] Hardy MH. The secret life of the hair follicle. [J]Trends Genet 1992;8:55- 61.
[15] 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. [J] Cell 1990;61:1329-37.
[16] Lyle S, Christofidou-Solomidou M, Liu Y, Elder DE, Albelda S, Cotsarelis G. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. [J] J Cell Sci 1998; 111:3179-88.
[17] Rochat A, Kobayashi K, Barrandon Y. Location of stem cells of human hair follicles by clonal analysis. [J]Cell 1994;76:1063-73.
[18] Danilenko DM, Ring BD, Pierce GF. Growth factors and cytokines in hair follicle development and cycling: recent insights from animal models and the potentials for clinical therapy. [J]Mol Med Today 1996;2:460-7.
[19] McGowan K, Coulombe PA. The wound repair-associated keratins 6, 16, and 17: insights into the role of intermediate filaments in specifying keratinocyte cytoarchitecture. [J]Subcell Biochem 1998;31:173-204.
[20] Staricco RG. Amelanotic melanocytes in the outer sheath of the humanhair follicle and their role in the repigmentation of regenerated epidermis. [J]Ann N Y Acad Sci 1963;100:239-55.
[21] Gilliam AC, Kremer IB, Yoshida Y, et al. The human hair follicle: a reservoir of CD40+ B7-deficient Langerhans cells that repopulate epidermis after UVB e×posure. [J]J Invest Dermatol 1998;110:422-7.
[22] Kim DK, Holbrook KA. The appearance, density, and distribution of Merkel cells in human embryonic and fetal skin: their relation to sweat gland and hair follicle development. [J]J Invest Dermatol 1995;104:411-6.
[23] Lindner G, Botchkarev VA, Botchkareva NV. Analysis of apoptosis during hair follicle regression. [J]Am J Pathol1997;151:1601-17.
[24] Slominski A, Paus R, Plonka P, et al. Melanogenesis during the anagen-catagen-telogen transformation of the murine hair cycle. [J] J Invest Dermatol 1994;102:862-9.
[25] Ahmad W, Faiyaz ul Haque M, Brancolini V, et al. Alopecia universalis associated with a mutation in the human hairless gene. [J] Science 1998;279:720-4.
[26] Dawber R, ed. Diseases of the hair and scalp. 3rd ed. Oxford, England:Blackwell Science, 1997.
[27] Ghazizadeh S, Taichman LB. Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin. [J]EMBO J 2001;20:1215–22.
[28] James M. Waters, Gavin D. Richardson, Colin A.B.Jahoda. Hair follicle stem cells. [J]seminars in cell & developmental biology,2007,18:245-254
[29] Blanpain C, Fuchs E. Epidermal stem cells of the skin. [J]Annu Rev Cell Dev Biol 2006;22:339–73.
[30] Reynolds AJ, Jahoda CA. Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis. [J]Development 1992;115:587–93.
[31] Oliver RF. Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. [J]J Embryol E×p Morphol 1966;15:331–47.
[32] Inamatsu M, Tochio T, Makabe A, Endo T, Oomizu S, Kobayashi E, et al. Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms. [J]Dev Growth Differ 2006;48:73–86.
[33] Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. [J]Exp Dermatol 2003;12: 126–36.
[34] Barry FP, Boynton RE, Haynesworth S, et al. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin(CD105). [J]Biochem Biophys Res Commun 1999;265 (1):134-139.
[35] Barry F, Boynton R, Murphy M, et al. The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. [J]Biochem Biophys Res Commun 2001;289(2):519-524.
[36] Haynesworth SE, Baber MA, Caplan AI. Cytokine e×pression by human marrow-derived mesenchymal progenitor cell in vitro: effects of dexamethasone and IL-1 alpha. [J] Cell Physiol 1996;166(3):585-592.
[37] Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesen- chymal stem cells from bone marrow. [J]Blood 2007;109(4):1743-1751.
[38] Conget PA, Minguell JJ. Phenotypical and functional properties of human bonemarrow mesenchymal progenitor cells. [J]Cell Physiol 1999;181(1): 67-73.
[39] AMOH Y, Lingna LI, Kensei KATSUOKA. Multipotent nestin-e×pressing hair follicle stem cells. [J] Journal of Dermatology 2009,36: 1–9
[40] Colin A B, Jahoda, Claire J, et al.Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages. [J]Experimental Dermatology 2003: 12: 849–859
[2] Seeberger KL, Dufour JM, Shapiro AM, et al. Expansion of mesenchymal stem cells from human pancreatic ductal epithelium. [ J ] Lab Invest 2006; 86(2):141-153.
[3] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. [J]Natrue 2002; 418(6893):41-49.
[4] Amoh Y, Li L, Katsuoka K, et al. Multipotentnestin positive, keratin-negative hair-follicle bulge stem cells can form neurons. [J]PNAS, 2005, 102(15):5530-5534.
[5] Lavker R, Sun T, Oshima H. et al. Hair follicle stem cells. J. Invest. Dermatol. Symp. Proc. 8, 28-38. (2003).
[6] Millar, S. E. Molecular mechanisms regulating hair follicle development. J. Invest. Dermatol. 2002,118, 216-225.
[7] Muller R, Handjiski B, vanderVeen. et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J. Invest. Dermatol. 117, 3-15.
[8] R Paus, G Cotsarelis .The Biology of Hair Follicles. [J]The New England Journal of Medicine.1999,Volume 341:491-497
[9] SchmidtUllrich, R. Paus, R. Molecular principles of hair follicle induction and morphogenesis. [J]BioEssays.2005 ,27, 247-261.
[10] Jahoda CA., Horne KA Oliver RF. Induction of hair growth by implantation of cultured dermal papilla cells. [J]Nature.1984 ,311, 560-562.
[11] Kishimoto J, Burgeson RE and Morgan BA. Wnt signaling maintains the hair-inducing activity of the dermal papilla. [J]Genes Dev. 2000 ,14, 1181-1185.
[12] Alonso L, Fuchs E.The hair cycle. Journal of Cell Science .2006, 119, 391-393
[13] Chuong CM. Molecular basis of epithelial appendage morphogenesis. [D]Molecular biology intelligence unit 1. Austin, Tex.: R.G. Landes, 1998.
[14] Hardy MH. The secret life of the hair follicle. [J]Trends Genet 1992;8:55- 61.
[15] 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. [J] Cell 1990;61:1329-37.
[16] Lyle S, Christofidou-Solomidou M, Liu Y, Elder DE, Albelda S, Cotsarelis G. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. [J] J Cell Sci 1998; 111:3179-88.
[17] Rochat A, Kobayashi K, Barrandon Y. Location of stem cells of human hair follicles by clonal analysis. [J]Cell 1994;76:1063-73.
[18] Danilenko DM, Ring BD, Pierce GF. Growth factors and cytokines in hair follicle development and cycling: recent insights from animal models and the potentials for clinical therapy. [J]Mol Med Today 1996;2:460-7.
[19] McGowan K, Coulombe PA. The wound repair-associated keratins 6, 16, and 17: insights into the role of intermediate filaments in specifying keratinocyte cytoarchitecture. [J]Subcell Biochem 1998;31:173-204.
[20] Staricco RG. Amelanotic melanocytes in the outer sheath of the humanhair follicle and their role in the repigmentation of regenerated epidermis. [J]Ann N Y Acad Sci 1963;100:239-55.
[21] Gilliam AC, Kremer IB, Yoshida Y, et al. The human hair follicle: a reservoir of CD40+ B7-deficient Langerhans cells that repopulate epidermis after UVB e×posure. [J]J Invest Dermatol 1998;110:422-7.
[22] Kim DK, Holbrook KA. The appearance, density, and distribution of Merkel cells in human embryonic and fetal skin: their relation to sweat gland and hair follicle development. [J]J Invest Dermatol 1995;104:411-6.
[23] Lindner G, Botchkarev VA, Botchkareva NV. Analysis of apoptosis during hair follicle regression. [J]Am J Pathol1997;151:1601-17.
[24] Slominski A, Paus R, Plonka P, et al. Melanogenesis during the anagen-catagen-telogen transformation of the murine hair cycle. [J] J Invest Dermatol 1994;102:862-9.
[25] Ahmad W, Faiyaz ul Haque M, Brancolini V, et al. Alopecia universalis associated with a mutation in the human hairless gene. [J] Science 1998;279:720-4.
[26] Dawber R, ed. Diseases of the hair and scalp. 3rd ed. Oxford, England:Blackwell Science, 1997.
[27] Ghazizadeh S, Taichman LB. Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin. [J]EMBO J 2001;20:1215–22.
[28] James M. Waters, Gavin D. Richardson, Colin A.B.Jahoda. Hair follicle stem cells. [J]seminars in cell & developmental biology,2007,18:245-254
[29] Blanpain C, Fuchs E. Epidermal stem cells of the skin. [J]Annu Rev Cell Dev Biol 2006;22:339–73.
[30] Reynolds AJ, Jahoda CA. Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis. [J]Development 1992;115:587–93.
[31] Oliver RF. Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. [J]J Embryol E×p Morphol 1966;15:331–47.
[32] Inamatsu M, Tochio T, Makabe A, Endo T, Oomizu S, Kobayashi E, et al. Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms. [J]Dev Growth Differ 2006;48:73–86.
[33] Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. [J]Exp Dermatol 2003;12: 126–36.
[34] Barry FP, Boynton RE, Haynesworth S, et al. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin(CD105). [J]Biochem Biophys Res Commun 1999;265 (1):134-139.
[35] Barry F, Boynton R, Murphy M, et al. The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. [J]Biochem Biophys Res Commun 2001;289(2):519-524.
[36] Haynesworth SE, Baber MA, Caplan AI. Cytokine e×pression by human marrow-derived mesenchymal progenitor cell in vitro: effects of dexamethasone and IL-1 alpha. [J] Cell Physiol 1996;166(3):585-592.
[37] Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesen- chymal stem cells from bone marrow. [J]Blood 2007;109(4):1743-1751.
[38] Conget PA, Minguell JJ. Phenotypical and functional properties of human bonemarrow mesenchymal progenitor cells. [J]Cell Physiol 1999;181(1): 67-73.
[39] AMOH Y, Lingna LI, Kensei KATSUOKA. Multipotent nestin-e×pressing hair follicle stem cells. [J] Journal of Dermatology 2009,36: 1–9
[40] Colin A B, Jahoda, Claire J, et al.Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages. [J]Experimental Dermatology 2003: 12: 849–859