绵羊胚胎成纤维细胞饲养层用于培养人诱导性多能干细胞的效果研究

doi:10.16431/j.cnki.1671-7236.2020.03.010

Abstract

Abstract: The aim of this study was to investigate the feasibility of using the isolated and mitomycin C-treated sheep embryonic fibroblast (SEF) in vitro as the feeder layer of the induced pluripotent stem cells (iPSCs).Taking SNL feeder layer cells as the control group and the human iPSC (hiPSC) as the experimental object,the effects of SEF cells and SNL cells as feeder layer of stem cells were compared by the morphological observation,the alkaline phosphatase (AP) staining,and the qRT-PCR and immunocytochemistry detection of the mRNA and protein expression for hiPSC marker genes.The results demonstrated that,being similar to hiPSC on SNL feeder layer,hiPSC on SEF feeder layer showed rapid growth and proliferation as colonies during the experimental period.Meanwhile,the AP staining was blue-purple,the undifferentiated state was maintained,and the pluripotent marker genes were normally expressed.The expression of hiPSC pluripotent marker genes c-myc,Klf4,OCT4,and SOX2 mRNAs,as well as the expression of OCT4,SOX2,SSEA4,and TRA-1-60 proteins were not significantly different for hiPSCs on SEF and SNL (P>0.05).The results indicated that SEF could be used as feeder layer cells for culturing iPSC in vitro,which laid a foundation for the further establishment of the gene-modified SEF cell lines that could express the growth-promoting factors.

Key words: sheep embryonic fibroblast(SEF); feeder layer; induced pluripotent stem cells(iPSC); pluripotency

CLC Number:

ZHANG Shiqiang, LI Mengxin, HAN Gaolian, GUO Lirong, ZHAO Dipeng, LI Junling, GUO Tianyan, DU Rong, QIN Jian. Research on the Effect of Sheep Embryonic Fibroblast Feeder for Culturing Human Induced Pluripotent Stem Cell[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 729-735.

References

[1] TAKAHASHI K,YAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126(4):663-676.
[2] TAKAHASHI K,TANABE K,Ohnuki M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,2007,131(5):861-872.
[3] YANG X,PABONL,MURRY C E.Engineering adolescence:Maturation of human pluripotent stem cell-derived cardiomyocytes[J].Circulation Research,2014,114(3):511-523.
[4] LEI W,FENG T,FANG X,et al.Signature of circular RNAs in human induced pluripotent stem cells and derived cardiomyocytes[J].Stem Cell Research & Therapy,2018,9(1):56.
[5] 鲁娣,张崴.胚胎干细胞及诱导多能干细胞在胚胎毒性研究中的应用[J].国际生殖健康/计划生育杂志,2017,36(2):130-136. LU D,ZHANG W.Application progress of embryonic stem cell and induced pluripotent stem cell in embryonic toxicity test[J].Journal of International Reproductive Health/Family Planning, 2017,36(2):130-136.(in Chinese)
[6] 樊亚男,关伟军,刘学东.干细胞治疗1型糖尿病研究进展[J].中国畜牧兽医,2015,42(11):3099-3104. FAN Y N,GUAN W J,LIU X D.Research progress on stem cell therapy for type 1 diabetes mellitus[J].China Animal Husbandry & Veterinary Medicine,2015,42(11):3099-3104.(in Chinese)
[7] 郭诗萌,刘世超,刘忠华.猪胚胎发育与胚胎工程研究进展[J].中国畜牧杂志,2017,53(9):1-3. GUO S M,LIU S C,LIU Z H.Research progress of the embryonic development and engineering in pig[J].Chinese Journal of Animal Science,2017,53(9):1-3.(in Chinese)
[8] 刘莹.浅谈猪诱导性多潜能干细胞的应用前景[J].中国畜牧兽医,2015,31(9):71-71. LIU Y.Application prospect of pig induced pluripotent stem cells[J].China Animal Husbandry & Veterinary Medicine,2015,31(9):71-71.(in Chinese)
[9] 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.
[10] REN Y,MA Z,YU T,et al.Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro[J].Scientific Reports,2018,8(1):7780.
[11] TAN E P,LI Y,VELASCOHERRERA M C,et al.Off-target assessment of CRISPR-Cas9 guiding RNAs in human iPS and mouse ES cells.[J].Genesis,2015,53(2):225-236.
[12] EVANS M J,KAUFMAN M H.Establishment in culture of pluripotential cells from mouse embryos[J].Nature,1981,292(5819):154-156.
[13] YANG J,RYAN D J,WANG W,et al.Establishment of mouse expanded potential stem cells[J].Nature,2017,550(7676):393-397.
[14] YANG Y,LIU B,XU J,et al.Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency[J].Cell,2017,169(2):243-257.
[15] KAUR J,TILKINS M L,ECKERT R,et al.Methods for culturing human embryonic stem cells in a xeno-free system[J].Methods in Molecular Biology,2013,997:115-126.
[16] YE J,BATES N,SOTREIOU D,et al.High quality clinical grade human embryonic stem cell lines derived from fresh discarded embryos[J].Stem Cell Research & Therapy,2017,8(1):128.
[17] CHEN G,GULBRANSON D R,HOU Z,et al.Chemically defined conditions for human iPSC derivation and culture[J].Nature Methods,2011,8(5):424-429.
[18] 邹灵秀,邓彦飞,陈凤,等.水牛转录因子Sox2和Lin28克隆与表达研究[J].中国畜牧杂志,2015,51(19):59-63. ZOU L X,DENG Y F,CHEN F,et al.Cloning and expression of buffalo transcription factors Sox2 and Lin28[J].Chinese Journal of Animal Science,2015,51(19):59-63.(in Chinese)
[19] LI Y,MCCLINTICK J,ZHONG L,et al.Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4[J].Blood,2005,105(2):635-637.
[20] ZAEHERS H,LENSCH M W,DAHERON L,et al.High-efficiency RNA interference in human embryonic stem cells[J].Stem Cells, 2005,23(3):299-305.
[21] MASUI S,NAKATAKE Y,TOYOOKA Y,et al.Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells[J].Nature Cell Biology,2007,9(6):625-635.
[22] 谢峰,张文杰,王鸿,等.SNL细胞作为饲养层细胞培养人胚胎干细胞[J].上海交通大学学报(医学版),2007,27(7):802-804. XIE F,ZHANG W J,WANG H,et al.SNL cells as new feeder cells to culture human embryonic cells[J].Journal of Shanghai Jiaotong University (Medical Science),2007,27(7):802-804.(in Chinese)
[23] OTA A,MATSUMURA K,LEE J J,et al.Stem Cell Keep^TM is effective for cryopreservation of human embryonic stem cells by vitrification[J].Cell Transplantation,2017,26(5):773-787.

Research on the Effect of Sheep Embryonic Fibroblast Feeder for Culturing Human Induced Pluripotent Stem Cell

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