昆明白小鼠胚胎生殖细胞的分离与培养
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摘要
胚胎生殖细胞(embryonic germ cells,EG)是来源于原始生殖细胞(primordial germ cells,PGC)的多潜能干细胞。本研究旨在优化昆明白小鼠EG细胞的培养条件。以昆明白小鼠10.5dpc胚胎PGC为材料,以丝裂霉素C处理的小鼠成纤维细胞(mouse embryonic germ cells,MEF)为饲养层,培养小鼠EG细胞,以对影响小鼠EG细胞传代的因素进行探讨。用ELISA试剂盒定量检测MEF自身分泌生长因子SCF、bFGF、LIF的浓度;以培养液是否添加生长因子,观察PGC/EG的培养效果;比较了机械分割法、胰酶消化法和胶原酶Ⅳ消化法对EG传代培养的影响;对获得的EG进行多能性鉴定。结果表明,培养3d的饲养层上清液中含75.7ng/L SCF、125.7ng/L bFGF、196.6ng/L LIF,培养5d的饲养层上清液中含76.2ng/L SCF、136.2ng/L bFGF、214.2ng/L LIF;培养液添加生长因子显著提高了EG的克隆数、传代数(P<0.05);机械分割法与酶消化法对EG传代差异显著(P<0.05),而两种酶法对EG传代无显著影响(P>0.05);所分离的EG细胞显示AKP染色强阳性;EG体外培养时会自分化为神经样细胞、上皮样细胞;EG细胞呈Oct4、SSEA-1的免疫荧光检测阳性。结果提示,饲养层自身能分泌少量生长因子,但培养液中添加生长因子可显著改善昆明白小鼠EG培养效果,酶消化法更适合于昆明白小鼠EG的传代培养。
Embryonic germ cells(EG)are pluripotent stem cells derived from primordial germ cells(PGC).The goal of this study was to optimize in vitro culture conditions for EG from Kunming White mice.Mitomycin C treated mouse embryonic fibroblasts(MEF)were used as feeder cells.Firstly,the amounts of SCF,bFGF,LIFsecreted by MEFwere measured by ELISA;Then,the proliferation of PGC/EG in presence of growth factors(SCF,bFGF,LIF)or not were measured;EG were passaged with mechanical segmentation or enzymatic dissociation to determine the most suitable passage method;Finally,the pluripotency of EG under different culture conditions were identified.The results showed,after 3 days in vitro culture,the supernatant of MEF contained 75.7 ng/L SCF,125.7 ng/LbFGF,196.6 ng/L LIF,respectively;While at day 5,the supernatant of MEF contained 76.2 ng/L SCF,136.2 ng/L bFGF,214.2 ng/L LIF;Addition of growth factors significantly increased EG colony numbers and passage numbers(P<0.05);Mechanical segmentation and enzymatic dissociation showed significant different effects on EG passage(P<0.05),while different enzymes(0.25%trypsin+0.04% EDTA or 0.1% collagenase IV)had no difference on EG passage(P>0.05);The cultured EG showed strong AKP activity,spontaneously differentiated into neuroblast-like and epithelial-like cells,and positive staining for Oct4 and SSEA-1 immunofluorescence identification.In conclusion,the feeders can secrete little growth factors,and the culture effects of EG is much better when adding growth factors to the culture medium.The enzymatic dissociation is more suitable for Kunming White mice EG passage.
Embryonic germ cells(EG)are pluripotent stem cells derived from primordial germ cells(PGC).The goal of this study was to optimize in vitro culture conditions for EG from Kunming White mice.Mitomycin C treated mouse embryonic fibroblasts(MEF)were used as feeder cells.Firstly,the amounts of SCF,bFGF,LIFsecreted by MEFwere measured by ELISA;Then,the proliferation of PGC/EG in presence of growth factors(SCF,bFGF,LIF)or not were measured;EG were passaged with mechanical segmentation or enzymatic dissociation to determine the most suitable passage method;Finally,the pluripotency of EG under different culture conditions were identified.The results showed,after 3 days in vitro culture,the supernatant of MEF contained 75.7 ng/L SCF,125.7 ng/LbFGF,196.6 ng/L LIF,respectively;While at day 5,the supernatant of MEF contained 76.2 ng/L SCF,136.2 ng/L bFGF,214.2 ng/L LIF;Addition of growth factors significantly increased EG colony numbers and passage numbers(P<0.05);Mechanical segmentation and enzymatic dissociation showed significant different effects on EG passage(P<0.05),while different enzymes(0.25%trypsin+0.04% EDTA or 0.1% collagenase IV)had no difference on EG passage(P>0.05);The cultured EG showed strong AKP activity,spontaneously differentiated into neuroblast-like and epithelial-like cells,and positive staining for Oct4 and SSEA-1 immunofluorescence identification.In conclusion,the feeders can secrete little growth factors,and the culture effects of EG is much better when adding growth factors to the culture medium.The enzymatic dissociation is more suitable for Kunming White mice EG passage.
引文
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[8]金立方.小鼠胚胎干细胞和胚胎生殖细胞的分离培养[D].陕西杨凌:西北农林科技大学,2004.
[9]Dong X,LI Y,Wang H J.Optimization of culture conditions for porcine embryonic germ cells[J].In Vitro Cellular&Developmental Biology-Animal,2016,52:131-136
[10]Cong Y M,Ma J,Sun R Z,et al.Derivation of putative porcine embryonic germ cells and analysis of their multi-lineage differentiation potential[J].Journal of Genetics and Genomics,2013,40:453-464
[11]Eiselleova L,Peterkova I,Neradil J,et al.Comparative study of mouse and human feeder cells for human embryonic stem cells[J].International Journal of Developmental Biology,2008,52(4):353-363
[12]Robert L.Handbook of Stem Cells(Volume 1):Embryonic Stem Cells[M].Northland:Elsevier Academic Press,2004,413-417
[13]Chen L R,Shiue YL,Bertolini L,et al.Establishment of pluripotent cell lines from porcine preimplantation embryos[J].Theriogenology,1999,52(2):195-212
[14]De Felici M,Pesce M,Giustiniani Q,et al.In vitro adhesiveness of mouse primordial germ cells to cellular and extracellular matrix component substrata[J].Microsc Res Tech,1998,43(3):258-264
[15]Shiue YL,Yang JR,Liao YJ,et al.Derivation of porcine pluripotent stem cells for biomedical research[J].Theriogenology,2016,86:176-181
[16]Petkov SG,Marks H,Klein T,et al.In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20-24of gestation[J].Stem cell research,2011,6(3):226-37
[17]Lee CK,Piedrahita JA.Effects of growth factors and feeder cells on porcine primordial germ cells in vitro[J].Cloning,2000,2(4):197-205
[18]Turnpenny L,Brickwood S,Spalluto CM,et al.Derivation of human embryonic germ cells:an alternative source of pluripotent stem cells[J].Stem Cells,2003,21(5):598-609
[19]Petkov SG,Anderson GB.Culture of porcine embryonic germ cells in serum-supplemented and serum-free conditions:the effects of serum and growth factors on primary and long-term culture[J].Cloning and stem cells,2008,10(2):263-76
[20]Shim H,Gutiérrez-Adán A,Chen LR,et al.Isolation of pluripotent stem cells from cultured porcine primordial germ cells[J].Biology of reproduction,1997,57(5):1089-95
[2]Matsui Y,Zsebo K,Hogan BL.Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture[J].Cell,1992,70(5):841-847
[3]Resnick JL,Bixler LS,CHENG L Z,et al.Long-term proliferation of mouse primordial germ cells in culture[J].Nature,1992,359(6395):550-551
[4]许新,丛笑倩,严缘昌.小鼠原始生殖细胞建系过程及其分化特性的研究[J].实验生物学报,1999,3(3):251-263
[5]Labosky PA,Barlow DP,Hogan BL.Mouse embryonic germ(EG)cell lines transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2receptor(Igf2r)gene compared with embryonic stem(ES)cell lines[J].Development,1994,120(11):3197-3204
[6]Stewart CL,Gadi I,Bhatt H.Stem cells from primordial germ cells can reenter the germ line[J].Developmental Biology,1994,161(2):626-628
[7]李吉霞,王新庄,李权武.昆明白小鼠胚胎生殖细胞的分离与培养[J].中国兽医科技,2005,35(10):790-793
[8]金立方.小鼠胚胎干细胞和胚胎生殖细胞的分离培养[D].陕西杨凌:西北农林科技大学,2004.
[9]Dong X,LI Y,Wang H J.Optimization of culture conditions for porcine embryonic germ cells[J].In Vitro Cellular&Developmental Biology-Animal,2016,52:131-136
[10]Cong Y M,Ma J,Sun R Z,et al.Derivation of putative porcine embryonic germ cells and analysis of their multi-lineage differentiation potential[J].Journal of Genetics and Genomics,2013,40:453-464
[11]Eiselleova L,Peterkova I,Neradil J,et al.Comparative study of mouse and human feeder cells for human embryonic stem cells[J].International Journal of Developmental Biology,2008,52(4):353-363
[12]Robert L.Handbook of Stem Cells(Volume 1):Embryonic Stem Cells[M].Northland:Elsevier Academic Press,2004,413-417
[13]Chen L R,Shiue YL,Bertolini L,et al.Establishment of pluripotent cell lines from porcine preimplantation embryos[J].Theriogenology,1999,52(2):195-212
[14]De Felici M,Pesce M,Giustiniani Q,et al.In vitro adhesiveness of mouse primordial germ cells to cellular and extracellular matrix component substrata[J].Microsc Res Tech,1998,43(3):258-264
[15]Shiue YL,Yang JR,Liao YJ,et al.Derivation of porcine pluripotent stem cells for biomedical research[J].Theriogenology,2016,86:176-181
[16]Petkov SG,Marks H,Klein T,et al.In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at Days 20-24of gestation[J].Stem cell research,2011,6(3):226-37
[17]Lee CK,Piedrahita JA.Effects of growth factors and feeder cells on porcine primordial germ cells in vitro[J].Cloning,2000,2(4):197-205
[18]Turnpenny L,Brickwood S,Spalluto CM,et al.Derivation of human embryonic germ cells:an alternative source of pluripotent stem cells[J].Stem Cells,2003,21(5):598-609
[19]Petkov SG,Anderson GB.Culture of porcine embryonic germ cells in serum-supplemented and serum-free conditions:the effects of serum and growth factors on primary and long-term culture[J].Cloning and stem cells,2008,10(2):263-76
[20]Shim H,Gutiérrez-Adán A,Chen LR,et al.Isolation of pluripotent stem cells from cultured porcine primordial germ cells[J].Biology of reproduction,1997,57(5):1089-95